Processes for producing hydrocarbon products

ABSTRACT

The present invention relates to processes for producing industrial products such as hydrocarbon products from non-polar lipids in a vegetative plant part. Preferred industrial products include alkyl esters which may be blended with petroleum based fuels.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/283,728, filedMay 21, 2014, now allowed, which is a continuation of U.S. Ser. No.13/725,404, filed Dec. 21, 2012, now U.S. Pat. No. 8,735,111, issued May27, 2014, which claims the benefit of U.S. Provisional Application No.61/718,563, filed Oct. 25, 2012, and 61/580,590, filed Dec. 27, 2011,the contents of each of which are hereby incorporated by reference intheir entirety into this application.

REFERENCE TO A SEQUENCE LISTING

This application incorporates-by-reference nucleotide and/or amino acidsequences which are present in the file named“150603_2251_83668-AAA_Substitute_Sequence_Listing_AHCtxt,” which is1.22 megabytes in size, and which was created Jun. 3, 2015 in the IBM-PCmachine format, having an operating system compatibility withMS-Windows, which is contained in the text file filed Jun. 3, 2015 aspart of this application.

FIELD OF THE INVENTION

The present invention relates to methods of producing industrialproducts such as hydrocarbon products from lipids produced in plants,particularly in vegetative parts of plants, and in algae and othernon-human organisms. In particular, the present invention providesplants having an increased level of one or more non-polar lipids such astriacylglycerols and an increased total non-polar lipid content. In oneparticular embodiment, the present invention relates to any combinationof lipid handling enzymes, oil body proteins and/or transcriptionfactors regulating lipid biosynthesis to increase the level of one ormore non-polar lipids and/or the total non-polar lipid content and/ormono-unsaturated fatty acid content in plants or any part thereofincluding plant seed and/or leaves, algae and fungi. In an embodiment,the conversion of the lipid to the hydrocarbon products occurs inharvested plant vegetative parts to produce alkyl esters of the fattyacids which are suitable for use as a renewable biodiesel fuel.

BACKGROUND OF INVENTION

The majority of the world's energy, particularly for transportation, issupplied by petroleum derived fuels, which have a finite supply.Alternative sources which are renewable are needed, such as frombiologically produced oils.

Triacylglycerol Biosynthesis

Triaclyglycerols (TAG) constitute the major form of lipids in seeds andconsist of three acyl chains esterified to a glycerol backbone. Thefatty acids are synthesized in the plastid as acyl-acyl carrier protein(ACP) intermediates where they can undergo a first desaturationcatalyzed. This reaction is catalyzed by the stearoyl-ACP desaturase andyields oleic acid (C18:1^(Δ9)). Subsequently, the acyl chains aretransported to the cytosol and endoplasmic reticulum (ER) asacyl-Coenzyme (CoA) esters. Prior to entering the major TAG biosynthesispathway, also known as the Kennedy or glycerol-3-phosphate (G3P)pathway, the acyl chains are typically integrated into phospholipids ofthe ER membrane where they can undergo further desaturation. Two keyenzymes in the production of polyunsaturated fatty acids are themembrane-bound FAD2 and FAD3 desaturases which produce linoleic(C18:2^(Δ9,12)) and α-linolenic acid (C18:3^(Δ9,12,15)) respectively.

TAG biosynthesis via the Kennedy pathway consists of a series ofsubsequent acylations, each using acyl-CoA esters as the acyl-donor. Thefirst acylation step typically occurs at the sn1-position of the G3Pbackbone and is catalyzed by the glycerol-3-phosphate acyltransferase(sn1-GPAT). The product, sn1-lysophosphatidic acid (sn1-LPA) serves as asubstrate for the lysophosphatidic acid acyltransferase (LPAAT) whichcouples a second acyl chain at the sn2-position to form phosphatidicacid. PA is further dephosphorylated to diacylglycerol (DAG) by thephosphatidic acid phosphatase (PAP) thereby providing the substrate forthe final acylation step. Finally, a third acyl chain is esterified tothe sn3-position of DAG in a reaction catalyzed by the diacylglycerolacyltransferase (DGAT) to form TAG which accumulates in oil bodies. Asecond enzymatic reaction, phosphatidyl glycerol acyltransferase (PDAT),also results in the conversion of DAG to TAG. This reaction is unrelatedto DGAT and uses phospholipids as the acyl-donors.

To maximise yields for the commercial production of lipids, there is aneed for further means to increase the levels of lipids, particularlynon-polar lipids such as DAGs and TAGs, in transgenic organisms or partsthereof such as plants, seeds, leaves, algae and fungi. Attempts atincreasing neutral lipid yields in plants have mainly focused onindividual critical enzymatic steps involved in fatty acid biosynthesisor TAG assembly. These strategies, however, have resulted in modestincreases in seed or leaf oil content. Recent metabolic engineering workin the oleaginous yeast Yarrowia lipolytica has demonstrated that acombined approach of increasing glycerol-3-phosphate production andpreventing TAG breakdown via β-oxidation resulted in cumulativeincreases in the total lipid content (Dulermo et al., 2011).

Plant lipids such as seedoil triaclyglycerols (TAGs) have many uses, forexample, culinary uses (shortening, texture, flavor), industrial uses(in soaps, candles, perfumes, cosmetics, suitable as drying agents,insulators, lubricants) and provide nutritional value. There is alsogrowing interest in using plant lipids for the production of biofuel.

To maximise yields for the commercial biological production of lipids,there is a need for further means to increase the levels of lipids,particularly non-polar lipids such as DAGs and TAGs, in transgenicorganisms or parts thereof such as plants, seeds, leaves, algae andfungi.

SUMMARY OF THE INVENTION

The present inventors have demonstrated significant increases in thelipid content of organisms, particularly in the vegetative parts andseed of plants, by manipulation of both fatty acid biosynthesis andlipid assembly pathways. Various combinations of genes were used toachieve substantial increases in oil content, which is of greatsignificance for production of biofuels and other industrial productsderived from oil.

In a first aspect, the invention provides a process for producing anindustrial product from a vegetative plant part or non-human organism orpart thereof comprising high levels of non-polar lipid.

In an embodiment, the present invention provides a process for producingan industrial product, the process comprising the steps of:

i) obtaining a vegetative plant part having a total non-polar lipidcontent of at least 10% (w/w dry weight),

ii) either

-   -   a) converting at least some of the lipid in the vegetative plant        part of step i) to the industrial product by applying heat,        chemical, or enzymatic means, or any combination thereof, to the        lipid in situ in the vegetative plant part, or    -   b) physically processing the vegetative plant part of step i),        and subsequently or simultaneously converting at least some of        the lipid in the processed vegetative plant part to the        industrial product by applying heat, chemical, or enzymatic        means, or any combination thereof, to the lipid in the processed        vegetative plant part, and

iii) recovering the industrial product, thereby producing the industrialproduct.

In another embodiment, the invention provides a process for producing anindustrial product, the process comprising the steps of:

i) obtaining a vegetative plant part having a total non-polar lipidcontent of at least about 3%, preferably at least about 5% or at leastabout 7% (w/w dry weight),

ii) converting at least some of the lipid in situ in the vegetativeplant part to the industrial product by heat, chemical, or enzymaticmeans, or any combination thereof, and

iii) recovering the industrial product, thereby producing the industrialproduct.

In another embodiment, the process for producing an industrial productcomprises the steps of:

i) obtaining a vegetative plant part having a total non-polar lipidcontent of at least about 3%, preferably at least about 5% or at leastabout 7% (w/w dry weight),

ii) physically processing the vegetative plant part of step i),

iii) converting at least some of the lipid in the processed vegetativeplant part to the industrial product by applying heat, chemical, orenzymatic means, or any combination thereof, to the lipid in theprocessed vegetative plant part, and

iv) recovering the industrial product, thereby producing the industrialproduct.

In another embodiment, the process for producing an industrial productcomprises the steps of:

i) obtaining a non-human organism or a part thereof comprising one ormore exogenous polynucleotide(s), wherein each of the one or moreexogenous polynucleotide(s) is operably linked to a promoter which iscapable of directing expression of the polynucleotide in a non-humanorganism or a part thereof, and wherein the non-human organism or partthereof has an increased level of one or more non-polar lipids relativeto a corresponding non-human organism or a part thereof lacking the oneor more exogenous polynucleotide(s), and

ii) converting at least some of the lipid in situ in the non-humanorganism or part thereof to the industrial product by heat, chemical, orenzymatic means, or any combination thereof, and

iii) recovering the industrial product, thereby producing the industrialproduct.

In a further embodiment, the process for producing an industrial productcomprises the steps of:

i) obtaining a non-human organism or a part thereof comprising one ormore exogenous polynucleotides, wherein the non-human organism or partthereof has an increased level of one or more non-polar lipids relativeto a corresponding non-human organism or a part thereof lacking the oneor more exogenous polynucleotides,

ii) physically processing the non-human organism or part thereof of stepi),

iii) converting at least some of the lipid in the processed non-humanorganism or part thereof to the industrial product by applying heat,chemical, or enzymatic means, or any combination thereof, to the lipidin the processed non-human organism or part thereof, and

iv) recovering the industrial product,

thereby producing the industrial product.

In each of the above embodiments, it would be understood by a personskilled in the art that the converting step could be done simultaneouslywith or subsequent to the physical processing step.

In each of the above embodiments, the total non-polar lipid content ofthe vegetative plant part, or non-human organism or part thereof,preferably a plant leaf or part thereof, stem or tuber, is at leastabout 3%, more preferably at least about 5%, preferably at least about7%, more preferably at least about 10%, more preferably at least about11%, more preferably at least about 12%, more preferably at least about13%, more preferably at least about 14%, or more preferably at leastabout 15% (w/w dry weight). In a further preferred embodiment, the totalnon-polar lipid content is between 5% and 25%, between 7% and 25%,between 10% and 25%, between 12% and 25%, between 15% and 25%, between7% and 20%, between 10% and 20%, between 10% and 15%, between 15% and20%, between 20% and 25%, about 10%, about 11%, about 12%, about 13%,about 14%, about 15%, about 16%, about 17%, about 18%, about 20%, orabout 22%, each as a percentage of dry weight. In a particularlypreferred embodiment, the vegetative plant part is a leaf (or leaves) ora portion thereof. In a more preferred embodiment, the vegetative plantpart is a leaf portion having a surface area of at least 1 cm².

Furthermore, in each of the above embodiments, the total TAG content ofthe vegetative plant part, or non-human organism or part thereof,preferably a plant leaf or part thereof, stem or tuber, is at leastabout 3%, more preferably at least about 5%, preferably at least about7%, more preferably at least about 10%, more preferably at least about11%, more preferably at least about 12%, more preferably at least about13%, more preferably at least about 14%, more preferably at least about15%, or more preferably at least about 17% (w/w dry weight). In afurther preferred embodiment, the total TAG content is between 5% and30%, between 7% and 30%, between 10% and 30%, between 12% and 30%,between 15% and 30%, between 7% and 30%, between 10% and 30%, between20% and 28%, between 18% and 25%, between 22% and 30%, about 10%, about11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%,about 18%, about 20%, or about 22%, each as a percentage of dry weight.In a particularly preferred embodiment, the vegetative plant part is aleaf (or leaves) or a portion thereof. In a more preferred embodiment,the vegetative plant part is a leaf portion having a surface area of atleast 1 cm².

Furthermore, in each of the above embodiments, the total lipid contentof the vegetative plant part, or non-human organism or part thereof,preferably a plant leaf or part thereof, stem or tuber, is at leastabout 3%, more preferably at least about 5%, preferably at least about7%, more preferably at least about 10%, more preferably at least about11%, more preferably at least about 12%, more preferably at least about13%, more preferably at least about 14%, more preferably at least about15%, more preferably at least about 17% (w/w dry weight), morepreferably at least about 20%, more preferably at least about 25%. In afurther preferred embodiment, the total lipid content is between 5% and35%, between 7% and 35%, between 10% and 35%, between 12% and 35%,between 15% and 35%, between 7% and 35%, between 10% and 20%, between18% and 28%, between 20% and 28%, between 22% and 28%, about 10%, about11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%,about 18%, about 20%, about 22%, or about 25%, each as a percentage ofdry weight. Typically, the total lipid content of the vegetative plantpart, or non-human organism or part thereof is about 2-3% higher thanthe non-polar lipid content. In a particularly preferred embodiment, thevegetative plant part is a leaf (or leaves) or a portion thereof. In amore preferred embodiment, the vegetative plant part is a leaf portionhaving a surface area of at least 1 cm².

The industrial product may be a hydrocarbon product such as fatty acidesters, preferably fatty acid methyl esters and/or a fatty acid ethylesters, an alkane such as methane, ethane or a longer-chain alkane, amixture of longer chain alkanes, an alkene, a biofuel, carbon monoxideand/or hydrogen gas, a bioalcohol such as ethanol, propanol, or butanol,biochar, or a combination of carbon monoxide, hydrogen and biochar. Theindustrial product may be a mixture of any of these components, such asa mixture of alkanes, or alkanes and alkenes, preferably a mixture whichis predominantly (>50%) C4-C8 alkanes, or predominantly C6 to C10alkanes, or predominantly C6 to C8 alkanes. The industrial product isnot carbon dioxide and not water, although these molecules may beproduced in combination with the industrial product. The industrialproduct may be a gas at atmospheric pressure/room temperature, orpreferably, a liquid, or a solid such as biochar, or the process mayproduce a combination of a gas component, a liquid component and a solidcomponent such as carbon monoxide, hydrogen gas, alkanes and biochar,which may subsequently be separated. In an embodiment, the hydrocarbonproduct is predominantly fatty acid methyl esters. In an alternativeembodiment, the hydrocarbon product is a product other than fatty acidmethyl esters.

The industrial product may be an intermediate product, for example, aproduct comprising fatty acids, which can subsequently be converted to,for example, biofuel by, for example, trans-esterification to fatty acidesters.

Heat may be applied in the process, such as by pyrolysis, combustion,gasification, or together with enzymatic digestion (including anaerobicdigestion, composting, fermentation). Lower temperature gasificationtakes place at, for example, between about 700° C. to about 1000° C.Higher temperature gasification takes place at, for example, betweenabout 1200° C. to about 1600° C. Lower temperature pyrolysis (slowerpyrolysis), takes place at, for example, about 400° C., whereas highertemperature pyrolysis takes place at, for example, about 500° C.Mesophilic digestion takes place between, for example, about 20° C. andabout 40° C. Thermophilic digestion takes place from, for example, about50° C. to about 65° C.

Chemical means include, but are not limited to, catalytic cracking,anaerobic digestion, fermentation, composting and transesterification.In an embodiment, a chemical means uses a catalyst or mixture ofcatalysts, which may be applied together with heat. The process may usea homogeneous catalyst, a heterogeneous catalyst and/or an enzymaticcatalyst. In an embodiment, the catalyst is a transition metal catalyst,a molecular sieve type catalyst, an activated alumina catalyst or sodiumcarbonate. Catalysts include acid catalysts such as sulphuric acid, oralkali catalysts such as potassium or sodium hydroxide or otherhydroxides. The chemical means may comprise transesterification of fattyacids in the lipid, which process may use a homogeneous catalyst, aheterogeneous catalyst and/or an enzymatic catalyst. The conversion maycomprise pyrolysis, which applies heat and may apply chemical means, andmay use a transition metal catalyst, a molecular sieve type catalyst, anactivated alumina catalyst and/or sodium carbonate.

Enzymatic means include, but are not limited to, digestion bymicroorganisms in, for example, anaerobic digestion, fermentation orcomposting, or by recombinant enzymatic proteins.

The lipid that is converted to an industrial product in this aspect ofthe invention may be some, or all, of the non-polar lipid in thevegetative plant part or non-human organism or part thereof, orpreferably the conversion is of at least some of the non-polar lipid andat least some of the polar lipid, and more preferably essentially all ofthe lipid (both polar and non-polar) in the vegetative plant part ornon-human organism or part thereof is converted to the industrialproduct(s).

In an embodiment, the conversion of the lipid to the industrial productoccurs in situ without physical disruption of the vegetative plant partor non-human organism or part thereof. In this embodiment, thevegetative plant part or non-human organism or part thereof may first bedried, for example by the application of heat, or the vegetative plantpart or non-human organism or part thereof may be used essentially asharvested, without drying. In an alternative embodiment, the processcomprises a step of physically processing the vegetative plant part, orthe non-human organism or part thereof. The physical processing maycomprise one or more of rolling, pressing such as flaking, crushing orgrinding the vegetative plant part, non human organism or part thereof,which may be combined with drying of the vegetative plant part, or thenon-human organism or part thereof. For example, the vegetative plantpart, or non-human organism or part thereof may first be substantiallydried and then ground to a finer material, for ease of subsequentprocessing.

In an embodiment, the weight of the vegetative plant part, or thenon-human organism or part thereof used in the process is at least 1 kgor preferably at least 1 tonne (dry weight) of pooled vegetative plantparts, or the non-human organisms or parts thereof. The processes mayfurther comprise a first step of harvesting vegetative plant parts, forexample from at least 100 or 1000 plants grown in a field, to provide acollection of at least 1000 such vegetative plant parts, i.e., which areessentially identical. Preferably, the vegetative plant parts areharvested at a time when the yield of non-polar lipids are at theirhighest. In one embodiment, the vegetative plant parts are harvestedabout at the time of flowering. In another embodiment, the vegetativeplant parts are harvested from about at the time of flowering to aboutthe beginning of senescence. In another embodiment, the vegetative plantparts are harvested when the plants are at least about 1 month of age.

The process may or may not further comprise extracting some of thenon-polar lipid content of the vegetative plant part, or the non-humanorganism or part thereof prior to the conversion step. In an embodiment,the process further comprises steps of:

(a) extracting at least some of the non-polar lipid content of thevegetative plant part or the non-human organism or part thereof asnon-polar lipid, and

(b) recovering the extracted non-polar lipid,

wherein steps (a) and (b) are performed prior to the step of convertingat least some of the lipid in the vegetative plant part, or thenon-human organism or part thereof to the industrial product. Theproportion of non-polar lipid that is first extracted may be less than50%, or more than 50%, or preferably at least 75% of the total non-polarlipid in the vegetative plant part, or non-human organism or partthereof. In this embodiment, the extracted non-polar lipid comprisestriacylglycerols, wherein the triacylglycerols comprise at least 90%,preferably at least 95% of the extracted lipid. The extracted lipid mayitself be converted to an industrial product other than the lipiditself, for example by trans-esterification to fatty acid esters.

In a second aspect, the invention provides a process for producingextracted lipid from a non-human organism or a part thereof.

In an embodiment, the present invention provides a process for producingextracted lipid, the process comprising the steps of:

i) obtaining a non-human organism or a part thereof, wherein thenon-human organism or part thereof has a total non-polar lipid contentof at least about 3%, more preferably at least about 5%, preferably atleast about 7%, more preferably at least about 10%, more preferably atleast about 11%, more preferably at least about 12%, more preferably atleast about 13%, more preferably at least about 14%, or more preferablyat least about 15% (w/w dry weight or seed weight),

ii) extracting lipid from the non-human organism or part thereof, and

iii) recovering the extracted lipid,

thereby producing the extracted lipid, wherein one or more or all of thefollowing features apply:

(a) the non-human organism or a part thereof comprises one or moreexogenous polynucleotide(s) and an increased level of one or morenon-polar lipid(s) relative to a corresponding non-human organism or apart thereof, respectively, lacking the one or more exogenouspolynucleotide(s), wherein each of the one or more exogenouspolynucleotides is operably linked to a promoter which is capable ofdirecting expression of the polynucleotide in a non-human organism orpart thereof,

(b) the non-human organism is an alga selected from the group consistingof diatoms (bacillariophytes), green algae (chlorophytes), blue-greenalgae (cyanophytes), golden-brown algae (chrysophytes), haptophytes,brown algae and heterokont algae,

(c) the one or more non-polar lipid(s) comprise a fatty acid whichcomprises a hydroxyl group, an epoxy group, a cyclopropane group, adouble carbon-carbon bond, a triple carbon-carbon bond, conjugateddouble bonds, a branched chain such as a methylated or hydroxylatedbranched chain, or a combination of two or more thereof, or any of two,three, four, five or six of the aforementioned groups, bonds or branchedchains,

(d) the total fatty acid content in the non-polar lipid(s) comprises atleast 2% more oleic acid and/or at least 2% less palmitic acid than thenon-polar lipid(s) in the corresponding non-human organism or partthereof lacking the one or more exogenous polynucleotides of part (a),

(e) the non-polar lipid(s) comprise a modified level of total sterols,preferably free (non-esterified) sterols, steroyl esters, steroylglycosides, relative to the non-polar lipid(s) in the correspondingnon-human organism or part thereof lacking the one or more exogenouspolynucleotides of part (a),

(f) the non-polar lipid(s) comprise waxes and/or wax esters,

(g) the non-human organism or part thereof is one member of a pooledpopulation or collection of at least about 1000 such non-human organismsor parts thereof, respectively, from which the lipid is extracted.

In another embodiment, the invention provides a process for producingextracted lipid, the process comprising the steps of:

i) obtaining a non-human organism or a part thereof comprising one ormore exogenous polynucleotide(s) and an increased level of one or morenon-polar lipid(s) relative to a corresponding non-human organism or apart thereof, respectively, lacking the one or more exogenouspolynucleotide(s),

ii) extracting lipid from the non-human organism or part thereof, and

iii) recovering the extracted lipid, thereby producing the extractedlipid, wherein each of the one or more exogenous polynucleotides isoperably linked to a promoter which is capable of directing expressionof the polynucleotide in a non-human organism or part thereof, andwherein one or more or all of the following features apply:

(a) the one or more exogenous polynucleotide(s) comprise a firstexogenous polynucleotide which encodes an RNA or transcription factorpolypeptide that increases the expression of one or more glycolytic orfatty acid biosynthetic genes in a non-human organism or a part thereof,and a second exogenous polynucleotide which encodes an RNA orpolypeptide involved in biosynthesis of one or more non-polar lipids,

(b) if the non-human organism is a plant, a vegetative part of the planthas a total non-polar lipid content of at least about 3%, morepreferably at least about 5%, preferably at least about 7%, morepreferably at least about 10%, more preferably at least about 11%, morepreferably at least about 12%, more preferably at least about 13%, morepreferably at least about 14%, or more preferably at least about 15%(w/w dry weight),

(c) the non-human organism is an alga selected from the group consistingof diatoms (bacillariophytes), green algae (chlorophytes), blue-greenalgae (cyanophytes), golden-brown algae (chrysophytes), haptophytes,brown algae and heterokont algae,

(d) the one or more non-polar lipid(s) comprise a fatty acid whichcomprises a hydroxyl group, an epoxy group, a cyclopropane group, adouble carbon-carbon bond, a triple carbon-carbon bond, conjugateddouble bonds, a branched chain such as a methylated or hydroxylatedbranched chain, or a combination of two or more thereof, or any of two,three, four, five or six of the aforementioned groups, bonds or branchedchains,

(e) the total fatty acid content in the non-polar lipid(s) comprises atleast 2% more oleic acid and/or at least 2% less palmitic acid than thenon-polar lipid(s) in the corresponding non-human organism or partthereof lacking the one or more exogenous polynucleotides,

(f) the non-polar lipid(s) comprise a modified level of total sterols,preferably free (non-esterified) sterols, steroyl esters, steroylglycosides, relative to the non-polar lipid(s) in the correspondingnon-human organism or part thereof lacking the one or more exogenouspolynucleotides,

(g) the non-polar lipid(s) comprise waxes and/or wax esters,

(h) the non-human organism or part thereof is one member of a pooledpopulation or collection of at least 1000 such non-human organisms orparts thereof, respectively, from which the lipid is extracted.

In an embodiment of (b) above, the total non-polar lipid content isbetween 5% and 25%, between 7% and 25%, between 10% and 25%, between 12%and 25%, between 15% and 25%, between 7% and 20%, between 10% and 20%,about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about16%, about 17%, about 18%, about 20%, or about 22%, each as a percentageof dry weight.

In an embodiment, the non-human organism is an alga, or an organismsuitable for fermentation such as a fungus, or preferably a plant. Thepart of the non-human organism may be a seed, fruit, or a vegetativepart of a plant. In a preferred embodiment, the plant part is a leafportion having a surface area of at least 1 cm². In another preferredembodiment, the non-human organism is a plant, the part is a plant seedand the extracted lipid is seedoil. In a more preferred embodiment, theplant is from an oilseed species, which is used commercially or could beused commercially for oil production. The species may be selected from agroup consisting of a Acrocomia aculeata (macauba palm), Arabidopsisthaliana, Aracinis hypogaea (peanut), Astrocaryum murumuru (murumuru),Astrocaryum vulgare (tucumã), Attalea geraensis (Indaiá-rateiro),Attalea humilis (American oil palm), Attalea oleifera (andaiá), Attaleaphalerata (uricuri), Attalea speciosa (babassu), Avena sativa (oats),Beta vulgaris (sugar beet), Brassica sp. such as Brassica carinata,Brassica juncea, Brassica napobrassica, Brassica napus (canola),Camelina sativa (false flax), Cannabis sativa (hemp), Carthamustinctorius (safflower), Caryocar brasiliense (pequi), Cocos nucifera(Coconut), Crambe abyssinica (Abyssinian kale), Cucumis melo (melon),Elaeis guineensis (African palm), Glycine max (soybean), Gossypiumhirsutum (cotton), Helianthus sp. such as Helianthus annuus (sunflower),Hordeum vulgare (barley), Jatropha curcas (physic nut), Joannesiaprinceps (arara nut-tree), Lemna sp. (duckweed) such as Lemnaaequinoctialis, Lemna disperma, Lemna ecuadoriensis, Lemna gibba(swollen duckweed), Lemna japonica, Lemna minor, Lemna minuta, Lemnaobscura, Lemna paucicostata, Lemna perpusilla, Lemna tenera, Lemnatrisulca, Lemna turionifera, Lemna valdiviana, Lemna yungensis, Licaniarigida (oiticica), Linum usitatissimum (flax), Lupinus angustifolius(lupin), Mauritia flexuosa (buriti palm), Maximiliana maripa (inajapalm), Miscanthus sp. such as Miscanthus×giganteus and Miscanthussinensis, Nicotiana sp. (tabacco) such as Nicotiana tabacum or Nicotianabenthamiana, Oenocarpus bacaba (bacaba-do-azeite), Oenocarpus bataua(patauã), Oenocarpus distichus (bacaba-de-leque), Oryza sp. (rice) suchas Oryza sativa and Oryza glaberrima, Panicum virgatum (switchgrass),Paraqueiba paraensis (mari), Persea amencana (avocado), Pongamia pinnata(Indian beech), Populus trichocarpa, Ricinus communis (castor),Saccharum sp. (sugarcane), Sesamum indicum (sesame), Solanum tuberosum(potato), Sorghum sp. such as Sorghum bicolor, Sorghum vulgare,Theobroma grandiforum (cupuassu), Trifolium sp., Trithrinax brasiliensis(Brazilian needle palm), Triticum sp. (wheat) such as Triticum aestivumand Zea mays (corn). In an embodiment, the Brassica napus plant is ofthe variety Westar. In an alternative embodiment, if the plant isBrassica napus, it is of a variety or cultivar other than Westar. In anembodiment, the plant is of a species other than Arabidopsis thaliana.In another embodiment, the plant is of a species other than Nicotianatabacum. In another embodiment, the plant is of a species other thanNicotiana benthamiana. In one embodiment, the plant is a perennial, forexample, a switchgrass. Each of the features described for the plant ofthe second aspect can be applied mutatis mutandis to the vegetativeplant part of the first aspect.

In an embodiment, the non-human organism is an oleaginous fungus such asan oleaginous yeast.

In a preferred embodiment, the lipid is extracted without drying thenon-human organism or part thereof prior to the extraction. Theextracted lipid may subsequently be dried or fractionated to reduce itsmoisture content.

In further embodiments of this aspect, the invention provides a processfor producing extracted lipid from specific oilseed plants. In anembodiment, the invention provides a process for producing extractedcanola oil, the process comprising the steps of

-   -   i) obtaining canola seed comprising at least 45% seedoil on a        weight basis,    -   ii) extracting oil from the canola seed, and    -   iii) recovering the oil, wherein the recovered oil comprises at        least 90% (w/w) triacylglycerols (TAG),        thereby producing the canola oil. In a preferred embodiment, the        canola seed has an oil content on a weight basis of at least        46%, at least 47%, at least 48%, at least 49%, at least 50%, at        least 51%, at least 52%, at least 53%, at least 54%, at least        55% or at least 56%. The oil content is determinable by        measuring the amount of oil that is extracted from the seed,        which is threshed seed as commonly harvested, and calculated as        a percentage of the seed weight, i.e., % (w/w). Moisture content        of the canola seed is between 5% and 15%, and is preferably        about 8.5%. In an embodiment, the oleic acid content is between        about 58% and 62% of the total fatty acid in the canola oil,        preferably at least 63%, and the palmitic acid content is about        4% to about 6% of the total fatty acids in the canola oil.        Preferred canola oil has an iodine value of 110-120 and a        chlorophyll level of less than 30 ppm.

In another embodiment, the invention provides a process for producingextracted cornseed oil, the process comprising the steps of:

-   -   i) obtaining corn seed comprising at least 5% seedoil on a        weight basis,    -   ii) extracting oil from the corn seed, and    -   iii) recovering the oil, wherein the recovered oil comprises at        least 80%, preferably at least 85% or at least 90% (w/w)        triacylglycerols (TAG),        thereby producing the cornseed oil. In a preferred embodiment,        the corn seed has an oil content on a seed weight basis (w/w) of        at least 6%, at least 7%, at least 8%, at least 9%, at least        10%, at least 11%, at least 12% or at least 13%. The moisture        content of the cornseed is about 13% to about 17%, preferably        about 15%. Preferred corn oil comprises about 0.1% tocopherols.

In another embodiment, the invention provides a process for producingextracted soybean oil, the process comprising the steps of:

-   -   i) obtaining soybean seed comprising at least 20% seedoil on a        weight basis,    -   ii) extracting oil from the soybean seed, and    -   iii) recovering the oil, wherein the recovered oil comprises at        least 90% (w/w) triacylglycerols (TAG),        thereby producing the soybean oil. In a preferred embodiment,        the soybean seed has an oil content on a seed weight basis (w/w)        of at least 21%, at least 22%, at least 23%, at least 24%, at        least 25%, at least 26%, at least 27%, at least 28%, at least        29%, at least 30%, or at least 31%. In an embodiment, the oleic        acid content is between about 20% and about 25% of the total        fatty acid in the soybean oil, preferably at least 30%, the        linoleic acid content is between about 45% and about 57%,        preferably less than 45%, and the palmitic acid content is about        10% to about 15% of the total fatty acids in the soybean oil,        preferably less than 10%. Preferably the soybean seed has a        protein content of about 40% on a dry weight basis, and the        moisture content of the soybean seed is about 10% to about 16%,        preferably about 13%.

In another embodiment, the invention provides a process for producingextracted lupinseed oil, the process comprising the steps of:

-   -   i) obtaining lupin seed comprising at least 10% seedoil on a        weight basis,    -   ii) extracting oil from the lupin seed, and    -   iii) recovering the oil, wherein the recovered oil comprises at        least 90% (w/w) triacylglycerols (TAG),        thereby producing the lupinseed oil. In a preferred embodiment,        the lupin seed has an oil content on a seed weight basis (w/w)        of at least 11%, at least 12%, at least 13%, at least 14%, at        least 15%, or at least 16%.

In another embodiment, the invention provides a process for producingextracted peanut oil, the process comprising the steps of:

-   -   i) obtaining peanuts comprising at least 50% seedoil on a weight        basis,    -   ii) extracting oil from the peanuts, and    -   iii) recovering the oil, wherein the recovered oil comprises at        least 90% (w/w) triacylglycerols (TAG),        thereby producing the peanut oil. In a preferred embodiment, the        peanut seed (peanuts) have an oil content on a seed weight basis        (w/w) of at least 51%, at least 52%, at least 53%, at least 54%,        at least 55% or at least 56%. In an embodiment, the oleic acid        content is between about 38% and 59% of the total fatty acid in        the peanut oil, preferably at least 60%, and the palmitic acid        content is about 9% to about 13% of the total fatty acids in the        peanut oil, preferably less than 9%.

In another embodiment, the invention provides a process for producingextracted sunflower oil, the process comprising the steps of:

-   -   i) obtaining sunflower seed comprising at least 50% seedoil on a        weight basis,    -   ii) extracting oil from the sunflower seed, and    -   iii) recovering the oil, wherein the recovered oil comprises at        least 90% (w/w) triacylglycerols (TAG),        thereby producing the sunflower oil. In a preferred embodiment,        the sunflower seed have an oil content on a seed weight basis        (w/w) of at least 51%, at least 52%, at least 53%, at least 54%,        or at least 55%.

In another embodiment, the invention provides a process for producingextracted cottonseed oil, the process comprising the steps of:

-   -   i) obtaining cottonseed comprising at least 41% seedoil on a        weight basis,    -   ii) extracting oil from the cottonseed, and    -   iii) recovering the oil, wherein the recovered oil comprises at        least 90% (w/w) triacylglycerols (TAG),        thereby producing the cottonseed oil. In a preferred embodiment,        the cotton seed have an oil content on a seed weight basis (w/w)        of at least 42%, at least 43%, at least 44%, at least 45%, at        least 46%, at least 47%, at least 48%, at least 49%, or at least        50%. In an embodiment, the oleic acid content is between about        15% and 22% of the total fatty acid in the cotton oil,        preferably at least 22%, the linoleic acid content is between        about 45% and about 57%, preferably less than 45%, and the        palmitic acid content is about 20% to about 26% of the total        fatty acids in the cottonseed oil, preferably less than 18%. In        an embodiment, the cottonseed oil also contains cyclopropanated        fatty acids such as sterculic and malvalic acids, and may        contain small amounts of gossypol.

In another embodiment, the invention provides a process for producingextracted safflower oil, the process comprising the steps of:

-   -   i) obtaining safflower seed comprising at least 35% seedoil on a        weight basis,    -   ii) extracting oil from the safflower seed, and    -   iii) recovering the oil, wherein the recovered oil comprises at        least 90% (w/w) triacylglycerols (TAG),        thereby producing the safflower oil. In a preferred embodiment,        the safflower seed have an oil content on a seed weight basis        (w/w) of at least 36%, at least 37%, at least 38%, at least 39%,        at least 40%, at least 41%, at least 42%, at least 43%, at least        44%, or at least 45%.

In another embodiment, the invention provides a process for producingextracted flaxseed oil, the process comprising the steps of:

-   -   i) obtaining flax seed comprising at least 36% seedoil on a        weight basis,    -   ii) extracting oil from the flax seed, and    -   iii) recovering the oil, wherein the recovered oil comprises at        least 90% (w/w) triacylglycerols (TAG),        thereby producing the flaxseed oil. In a preferred embodiment,        the flax seed have an oil content on a seed weight basis (w/w)        of at least 37%, at least 38%, at least 39%, or at least 40%.

In another embodiment, the invention provides a process for producingextracted Camelina oil, the process comprising the steps of:

-   -   i) obtaining Camelina sativa seed comprising at least 36%        seedoil on a weight basis,    -   ii) extracting oil from the Camelina sativa seed, and    -   iii) recovering the oil, wherein the recovered oil comprises at        least 90% (w/w) triacylglycerols (TAG),        thereby producing the Camelina oil. In a preferred embodiment,        the Camelina sativa seed have an oil content on a seed weight        basis (w/w) of at least 37%, at least 38%, at least 39%, at        least 40%, at least 41%, at least 42%, at least 43%, at least        44%, or at least 45%.

The process of the second aspect may also comprise measuring the oiland/or protein content of the seed by near-infrared reflectancespectroscopy as described in Horn et al. (2007).

In an embodiment, the process of the second aspect of the inventioncomprises partially or completely drying the vegetative plant part, orthe non-human organism, or part thereof, or the seed, and/or one or moreof rolling, pressing such as flaking, crushing or grinding thevegetative plant part, or the non-human organism or part thereof, or theseed, or any combination of these methods, in the extraction process.The process may use an organic solvent (e.g., hexane such as n-hexane ora combination of n-hexane with isohexane, or butane alone or incombination with hexane) in the extraction process to extract the lipidor oil or to increase the efficiency of the extraction process,particularly in combination with a prior drying process to reduce themoisture content.

In an embodiment, the process comprises recovering the extracted lipidor oil by collecting it in a container, and/or purifying the extractedlipid or seedoil, such as, for example, by degumming, deodorising,decolourising, drying and/or fractionating the extracted lipid or oil,and/or removing at least some, preferably substantially all, waxesand/or wax esters from the extracted lipid or oil. The process maycomprise analysing the fatty acid composition of the extracted lipid oroil, such as, for example, by converting the fatty acids in theextracted lipid or oil to fatty acid methyl esters and analysing theseusing GC to determine the fatty acid composition. The fatty acidcomposition of the lipid or oil is determined prior to any fractionationof the lipid or oil that alters its fatty acid composition. Theextracted lipid or oil may comprise a mixture of lipid types and/or oneor more derivatives of the lipids, such as free fatty acids.

In an embodiment, the process of the second aspect of the inventionresults in substantial quantities of extracted lipid or oil. In anembodiment, the volume of the extracted lipid or oil is at least 1liter, preferably at least 10 liters. In a preferred embodiment, theextracted lipid or oil is packaged ready for transportation or sale.

In an embodiment, the extracted lipid or oil comprises at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% or at least 96% TAGon a weight basis. The extracted lipid or oil may comprise phospholipidas a minor component, up to about 8% by weight, preferably less than 5%by weight, and more preferably less than 3% by weight.

In an embodiment, the process results in extracted lipid or oil whereinone or more or all of the following features apply:

(i) triacylglycerols comprise at least 90%, preferably at least 95% or96%, of the extracted lipid or oil,

(ii) the extracted lipid or oil comprises free sterols, steroyl esters,steroyl glycosides, waxes or wax esters, or any combination thereof, and

(iii) the total sterol content and/or composition in the extracted lipidor oil is significantly different to the sterol content and/orcomposition in the extracted lipid or oil produced from a correspondingnon-human organism or part thereof, or seed.

In an embodiment, the process further comprises converting the extractedlipid or oil to an industrial product. That is, the extracted lipid oroil is converted post-extraction to another chemical form which is anindustrial product. Preferably, the industrial product is a hydrocarbonproduct such as fatty acid esters, preferably fatty acid methyl estersand/or fatty acid ethyl esters, an alkane such as methane, ethane or alonger-chain alkane, a mixture of longer chain alkanes, an alkene, abiofuel, carbon monoxide and/or hydrogen gas, a bioalcohol such asethanol, propanol, or butanol, biochar, or a combination of carbonmonoxide, hydrogen and biochar.

In the process of either the first or second aspects of the invention,the vegetative plant part, or the part of the non-human organism may bean aerial plant part or a green plant part such as a plant leaf or stem,a woody part such as a stem, branch or trunk, or a root or tuber.Preferably, the plants are grown in a field and the parts such as seedharvested from the plants in the field.

In an embodiment, the process further comprises a step of harvesting thevegetative plant part, non-human organism or part thereof, preferablywith a mechanical harvester.

Preferably, the vegetative plant parts are harvested at a time when theyield of non-polar lipids are at their highest. In one embodiment, thevegetative plant parts are harvested about at the time of flowering. Inanother embodiment, the vegetative plant parts are harvested from aboutat the time of flowering to about the beginning of senescence. Inanother embodiment, the vegetative plant parts are harvested when theplants are at least about 1 month of age.

If the organism is an algal or fungal organism, the cells may be grownin an enclosed container or in an open-air system such as a pond. Theresultant organisms comprising the non-polar lipid may be harvested,such as, for example, by a process comprising filtration,centrifugation, sedimentation, flotation or flocculation of algal orfungal organisms such as by adjusting pH of the medium. Sedimentation isless preferred.

In the process of the second aspect of the invention, the totalnon-polar lipid content of the non-human organism or part thereof, sucha vegetative plant part or seed, is increased relative to acorresponding vegetative plant part, non-human organism or part thereof,or seed.

In an embodiment, the vegetative plant part, or non-human organism orpart thereof, or seed of the first or second aspects of the invention isfurther defined by three features, namely Feature (i), Feature (ii) andFeature (iii), singly or in combination:

Feature (i) quantifies the extent of the increased level of the one ormore non-polar lipids or the total non-polar lipid content of thevegetative plant part, or non-human organism or part thereof, or seedwhich may be expressed as the extent of increase on a weight basis (dryweight basis or seed weight basis), or as the relative increase comparedto the level in the corresponding vegetative plant part, or non-humanorganism or part thereof, or seed. Feature (ii) specifies the plantgenus or species, or the fungal or algal species, or other cell type,and Feature (iii) specifies one or more specific lipids that areincreased in the non-polar lipid content.

For Feature (i), in an embodiment, the extent of the increase of the oneor more non-polar lipids is at least 0.5%, at least 1%, at least 2%, atleast 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%,at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, atleast 19%, at least 20%, at least 21%, at least 22%, at least 23%, atleast 24%, at least 25% or at least 26% greater on a dry weight or seedweight basis than the corresponding vegetative plant part, or non-humanorganism or part thereof.

Also for Feature (i), in a preferred embodiment, the total non-polarlipid content of the vegetative plant part, or non-human organism orpart thereof, or seed is increased when compared to the correspondingvegetative plant part, or non-human organism or part thereof, or seed.In an embodiment, the total non-polar lipid content is increased by atleast 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, atleast 11%, at least 12%, at least 13%, at least 14%, at least 15%, atleast 16%, at least 17%, at least 18%, at least 19%, at least 20%, atleast 21%, at least 22%, at least 23%, at least 24%, at least 25% or atleast 26% greater on a dry weight or seed weight basis than thecorresponding vegetative plant part, or non-human organism or partthereof, or seed.

Further, for Feature (i), in an embodiment, the level of the one or morenon-polar lipids and/or the total non-polar lipid content is at least1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, atleast 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least12%, at least 13%, at least 14%, at least 15%, at least 16%, at least17%, at least 18%, at least 19%, at least 20%, at least 21%, at least22%, at least 23%, at least 24%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, or at least 100% greater on a relativebasis than the corresponding vegetative plant part, or non-humanorganism or part thereof, or seed.

Also for Feature (i), the extent of increase in the level of the one ormore non-polar lipids and/or the total non-polar lipid content may be atleast 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, atleast 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, atleast 10-fold, or at least 12-fold, preferably at least about 13-fold orat least about 15-fold greater on a relative basis than thecorresponding vegetative plant part, or non-human organism or partthereof, or seed.

As a result of the increase in the level of the one or more non-polarlipids and/or the total non-polar lipid content as defined in Feature(i), the total non-polar lipid content of the vegetative plant part, ornon-human organism or part thereof, or seed is preferably between 5% and25%, between 7% and 25%, between 10% and 25%, between 12% and 25%,between 15% and 25%, between 7% and 20%, between 10% and 20%, about 10%,about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about17%, about 18%, about 20%, or about 22%, each as a percentage of dryweight or seed weight.

For Feature (ii), in an embodiment, the non-human organism is a plant,alga, or an organism suitable for fermentation such as a yeast or otherfungus, preferably an oleaginous fungus such as an oleaginous yeast. Theplant may be, or the vegetative plant part may be from, for example, aplant which is Acrocomia aculeata (macauba palm), Arabidopsis thaliana,Aracinis hypogaea (peanut), Astrocaryum murumuru (murumuru), Astrocaryumvulgare (tucumã), Attalea geraensis (Indaiá-rateiro), Attalea humilis(American oil palm), Attalea oleifera (andaiá), Attalea phalerata(uricuri), Attalea speciosa (babassu), Avena sativa (oats), Betavulgaris (sugar beet), Brassica sp. such as Brassica carinata, Brassicajuncea, Brassica napobrassica, Brassica napus (canola), Camelina sativa(false flax), Cannabis sativa (hemp), Carthamus tinctorius (safflower),Caryocar brasiliense (pequi), Cocos nucifera (Coconut), Crambeabyssinica (Abyssinian kale), Cucumis melo (melon), Elaeis guineensis(African palm), Glycine max (soybean), Gossypium hirsutum (cotton),Helianthus sp. such as Helianthus annuus (sunflower), Hordeum vulgare(barley), Jatropha curcas (physic nut), Joannesia princeps (araranut-tree), Lemna sp. (duckweed) such as Lemna aequinoctialis, Lemnadisperma, Lemna ecuadoriensis, Lemna gibba (swollen duckweed), Lemnajaponica, Lemna minor, Lemna minuta, Lemna obscura, Lemna paucicostata,Lemna perpusilla, Lemna tenera, Lemna trisulca, Lemna turionifera, Lemnavaldiviana, Lemna yungensis, Licania rigida (oiticica), Linumusitatissimum (flax), Lupines angustifolius (lupin), Mauritia flexuosa(buriti palm), Maximiliana maripa (inaja palm), Miscanthus sp. such asMiscanthus×giganteus and Miscanthus sinensis, Nicotiana sp. (tobacco)such as Nicotiana tabacum or Nicotiana benthamiana, Oenocarpus bacaba(bacaba-do-azeite), Oenocarpus bataua (patauã), Oenocarpus distichus(bacaba-de-leque), Oryza sp. (rice) such as Oryza sativa and Oryzaglaberrima, Panicum virgatum (switchgrass), Paraqueiba paraensis (mari),Persea amencana (avocado), Pongamia pinnata (Indian beech), Populustrichocarpa, Ricinus communis (castor), Saccharum sp. (sugarcane),Sesamum indicum (sesame), Solanum tuberosum (potato), Sorghum sp. suchas Sorghum bicolor, Sorghum vulgare, Theobroma grandiforum (cupuassu),Trifolium sp., Trithrinax brasiliensis (Brazilian needle palm), Triticumsp. (wheat) such as Triticum aestivum and Zea mays (corn). In anembodiment, the Brassica napus plant is of the variety Westar. In analternative embodiment, if the plant is Brassica napus, it is of avariety or cultivar other than Westar. In an embodiment, the plant is ofa species other than Arabidopsis thaliana. In another embodiment, theplant is of a species other than Nicotiana tabacum. In anotherembodiment, the plant is of a species other than Nicotiana benthamiana.In one embodiment, the plant is a perennial, for example, a switchgrass.Each of the features described for the plant of the second aspect can beapplied mutatis mutandis to the vegetative plant part of the firstaspect.

For Feature (iii), TAG, DAG, TAG and DAG, MAG, total polyunsaturatedfatty acid (PUFA), or a specific PUFA such as eicosadienoic acid (EDA),arachidonic acid (ARA), alpha linolenic acid (ALA), stearidonic acid(SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA),eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA),docosahexaenoic acid (DHA), or a fatty acid which comprises a hydroxylgroup, an epoxy group, a cyclopropane group, a double carbon-carbonbond, a triple carbon-carbon bond, conjugated double bonds, a branchedchain such as a methylated or hydroxylated branched chain, or acombination of two or more thereof, or any of two, three, four, five orsix of the aforementioned groups, bonds or branched chains, is/areincreased or decreased. The extent of the increase of TAG, DAG, TAG andDAG, MAG, PUFA, specific PUFA, or fatty acid, is as defined in Feature(i) above. In a preferred embodiment, the MAG is 2-MAG. Preferably, DAGand/or TAG, more preferably the total of DAG and TAG, or MAG and TAG,are increased. In an embodiment, TAG levels are increased withoutincreasing the MAG and/or DAG content.

Also for Feature (iii), in an embodiment, the total fatty acid contentand/or TAG content of the total non-polar lipid content comprises (a) atleast 2% more, preferably at least 5% more, more preferably at least 7%more, most preferably at least 10% more, at least 15% more, at least 20%more, at least 25% more oleic acid, or at least 30% more relative to thenon-polar lipid(s) in the corresponding vegetative plant part, ornon-human organism or part thereof, or seed lacking the one or moreexogenous polynucleotides. In an embodiment, the total fatty acidcontent in the non-polar lipid(s) comprises (b) at least 2% less,preferably at least 4% less, more preferably at least 7% less, at least10% less, at least 15% less, or at least 20% less palmitic acid relativeto the non-polar lipid(s) in the corresponding vegetative plant part, ornon-human organism or part thereof, or seed lacking the one or moreexogenous polynucleotides. In an embodiment, the total fatty acidcontent of the total non-polar lipid content comprises (c) at least 2%less, preferably at least 4% less, more preferably at least 7% less, atleast 10% less, or at least 15% less ALA relative to the non-polarlipid(s) in the corresponding vegetative plant part, or non-humanorganism or part thereof, or seed lacking the one or more exogenouspolynucleotides. In an embodiment, the total fatty acid content of thetotal non-polar lipid content comprises (d) at least 2% more, preferablyat least 5% more, more preferably at least 7% more, most preferably atleast 10% more, or at least 15% more, LA, relative to the non-polarlipid(s) in the corresponding vegetative plant part, or non-humanorganism or part thereof, or seed lacking the one or more exogenouspolynucleotides. Most preferably, the total fatty acid and/or TAGcontent of the total non-polar lipid content has an increased oleic acidlevel according to a figure defined in (a) and a decreased palmitic acidcontent according to a figure defined in (b). In an embodiment, thetotal sterol content is increased by at least 10% relative to seedoilfrom a corresponding seed. In an embodiment, the extracted lipid or oilcomprises at least 10 ppm chlorophyll, preferably at least 30 ppmchlorophyll. The chlorophyll may subsequently be removed byde-colourising the extracted lipid or oil.

In preferred embodiments, the one or more non-polar lipids and/or thetotal non-polar lipid content is defined by the combination of Features(i), (ii) and (iii), or Features (i) and (ii), or Features (i) and(iii), or Features (ii) and (iii).

The process of the second aspect of the invention provides, in anembodiment, that one or more or all of the following features apply:

(i) the level of one or more non-polar lipids in the vegetative plantpart, or non-human organism or part thereof, or seed is at least 0.5%greater on a weight basis than the level in a corresponding vegetativeplant part, non-human organism or part thereof, or seed, respectively,lacking the one or more exogenous polynucleotide(s), or preferably asfurther defined in Feature (i),

(ii) the level of one or more non-polar lipids in the vegetative plantpart, non-human organism or part thereof, or seed is at least 1% greateron a relative basis than in a corresponding vegetative plant part,non-human organism or part thereof, or seed, respectively, lacking theone or more exogenous polynucleotide(s), or preferably as furtherdefined in Feature (i),

(iii) the total non-polar lipid content in the vegetative plant part,non-human organism or part thereof, or seed is at least 0.5% greater ona weight basis than the level in a corresponding vegetative plant part,non-human organism or part thereof, or seed, respectively, lacking theone or more exogenous polynucleotide(s), or preferably as furtherdefined in Feature (i),

(iv) the total non-polar lipid content in the vegetative plant part,non-human organism or part thereof, or seed is at least 1% greater on arelative basis than in a corresponding vegetative plant part, non-humanorganism or part thereof, or seed, respectively, lacking the one or moreexogenous polynucleotide(s), or preferably as further defined in Feature(i),

(v) the level of one or more non-polar lipids and/or the total non-polarlipid content of the vegetative plant part, non-human organism or partthereof, or seed, is at least 0.5% greater on a weight basis and/or atleast 1% greater on a relative basis than a corresponding vegetativeplant part, non-human organism or a part thereof, or seed, respectively,which is lacking the one or more exogenous polynucleotides and whichcomprises an exogenous polynucleotide encoding an Arabidopsis thalianaDGAT1, or preferably as further defined in Feature (i),

(vi) the TAG, DAG, TAG and DAG, or MAG content in the lipid in thevegetative plant part, non-human organism or part thereof, or seed,and/or in the extracted lipid therefrom, is at least 10% greater on arelative basis than the TAG, DAG, TAG and DAG, or MAG content in thelipid in a corresponding vegetative plant part, non-human organism or apart thereof, or seed lacking the one or more exogenouspolynucleotide(s), or a corresponding extracted lipid therefrom,respectively, or preferably as further defined in Feature (i), and

(vii) the total polyunsaturated fatty acid (PUFA) content in the lipidin the vegetative plant part, non-human organism or part thereof, orseed and/or in the extracted lipid therefrom, is increased (e.g., in thepresence of a MGAT) or decreased (e.g., in the absence of a MGAT)relative to the total PUFA content in the lipid in a correspondingvegetative plant part, non-human organism or part thereof, or seedlacking the one or more exogenous polynucleotide(s), or a correspondingextracted lipid therefrom, respectively, or preferably as furtherdefined in Feature (i) or Feature (iii).

In an embodiment, the level of a PUFA in the vegetative plant part,non-human organism or part thereof, or seed and/or the extracted lipidtherefrom, is increased relative to the level of the PUFA in acorresponding vegetative plant part, non-human organism or part thereof,or seed, or a corresponding extracted lipid therefrom, respectively,wherein the polyunsaturated fatty acid is eicosadienoic acid,arachidonic acid (ARA), alpha linolenic acid (ALA), stearidonic acid(SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA),eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA),docosahexaenoic acid (DHA), or a combination of two of more thereof.Preferably, the extent of the increase is as defined in Feature (i).

In an embodiment of the second aspect, the corresponding vegetativeplant part, or non-human organism or part thereof, or seed is anon-transgenic vegetative plant part, or non-human organism or partthereof, or seed, respectively. In a preferred embodiment, thecorresponding vegetative plant part, or non-human organism or partthereof, or seed is of the same cultivar, strain or variety but lackingthe one or more exogenous polynucleotides. In a further preferredembodiment, the corresponding vegetative plant part, or non-humanorganism or part thereof, or seed is at the same developmental stage,for example, flowering, as the vegetative plant part, or non-humanorganism or part thereof, or seed. In another embodiment, the vegetativeplant parts are harvested from about at the time of flowering to aboutthe beginning of senescence. In another embodiment, the seed isharvested when the plants are at least about 1 month of age.

In an embodiment, part of the non-human organism is seed and the totaloil content, or the total fatty acid content, of the seed is at least0.5% to 25%, or at least 1.0% to 24%, greater on a weight basis than acorresponding seed lacking the one or more exogenous polynucleotides.

In an embodiment, the relative DAG content of the seedoil is at least10%, at least 10.5%, at least 11%, at least 11.5%, at least 12%, atleast 12.5%, at least 13%, at least 13.5%, at least 14%, at least 14.5%,at least 15%, at least 15.5%, at least 16%, at least 16.5%, at least17%, at least 17.5%, at least 18%, at least 18.5%, at least 19%, atleast 19.5%, at least 20% greater on a relative basis than of seedoilfrom a corresponding seed. In an embodiment, the DAG content of the seedis increased by an amount as defined in Feature (i) and the seed is froma genus and/or species as defined in Feature (ii).

In an embodiment, the relative TAG content of the seed is at least 5%,at least 5.5%, at least 6%, at least 6.5%, at least 7%, at least 7.5%,at least 8%, at least 8.5%, at least 9%, at least 9.5%, at least 10%, orat least 11% greater on an absolute basis relative to a correspondingseed. In an embodiment, the TAG content of the seed is increased by anamount as defined in Feature (i) and the seed is from a genus and/orspecies as defined in Feature (ii).

In another embodiment, the part of the non-human organism is avegetative plant part and the TAG, DAG, TAG and DAG, or MAG content ofthe vegetative plant part is at least 10%, at least 11%, at least 12%,at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, atleast 18%, at least 19%, at least 20%, at least 21%, at least 22%, atleast 23%, at least 24%, at least 25%, at least 30% at least 35%, atleast 40%, at least 45%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or at least 100% greater on a relative basisthan the TAG, DAG, TAG and DAG, or MAG content of a correspondingvegetative plant part lacking the one or more exogenous polynucleotides.In a preferred embodiment, the MAG is 2-MAG. In an embodiment, the TAG,DAG, TAG and DAG, or MAG content of the vegetative plant part isdetermined from the amount of these lipid components in the extractablelipid of the vegetative plant part. In a further embodiment, the TAG,DAG, TAG and DAG, or MAG content of the transgenic vegetative plant partis increased by an amount as defined in Feature (i).

In an embodiment, at least 20% (mol %), at least 22% (mol %), at least30% (mol %), at least 40% (mol %), at least 50% (mol %) or at least 60%(mol %), preferably at least 65% (mol %), more preferably at least 66%(mol %), at least 67% (mol %), at least 68% (mol %), at least 69% (mol%) or at least 70% (mol %) of the fatty acid content of the totalnon-polar lipid content of the vegetative plant part, non-human organismor part thereof, or seed, or of the lipid or oil extracted therefrom,preferably of the TAG fraction, is oleic acid. Such high oleic contentsare preferred for use in biodiesel applications.

In another embodiment, the PUFA content of the vegetative plant part, ornon-human organism or part thereof, or seed is increased (e.g., in thepresence of a MGAT) or decreased (e.g., in the absence of a MGAT) whencompared to the corresponding vegetative plant part, or non-humanorganism or part thereof, or seed. In this context, the PUFA contentincludes both esterified PUFA (including TAG, DAG, etc.) andnon-esterified PUFA. In an embodiment, the PUFA content of thevegetative plant part, or non-human organism or part thereof, or seed ispreferably determined from the amount of PUFA in the extractable lipidof the vegetative plant part, or non-human organism or part thereof, orseed. The extent of the increase in PUFA content may be as defined inFeature (i). The PUFA content may comprise EDA, ARA, ALA, SDA, ETE, ETA,EPA, DPA, DHA, or a combination of two of more thereof.

In another embodiment, the level of a PUFA in the vegetative plant part,non-human organism or part thereof, or seed, or the lipid or oilextracted therefrom is increased or decreased when compared to thecorresponding vegetative plant part, non-human organism or part thereof,or seed, or the lipid or oil extracted therefrom. The PUFA may be EDA,ARA, ALA, SDA, ETE, ETA, EPA, DPA, DHA, or a combination of two of morethereof. The extent of the increase in the PUFA may be as defined inFeature (i).

In another embodiment, the level of a fatty acid in the extracted lipidor oil is increased when compared to the lipid extracted from thecorresponding vegetative plant part, or non-human organism or partthereof, or seed and wherein the fatty acid comprises a hydroxyl group,an epoxy group, a cyclopropane group, a double carbon-carbon bond, atriple carbon-carbon bond, conjugated double bonds, a branched chainsuch as a methylated or hydroxylated branched chain, or a combination oftwo or more thereof, or any of two, three, four, five or six of theaforementioned groups, bonds or branched chains. The extent of theincrease in the fatty acid may be as defined in Feature (i).

In an embodiment, the level of the one or more non-polar lipids (such asTAG, DAG, TAG and DAG, MAG, PUFA, or a specific PUFA, or a specificfatty acid) and/or the total non-polar lipid content is determinable byanalysis by using gas chromatography of fatty acid methyl estersobtained from the extracted lipid. Alternate methods for determining anyof these contents are known in the art, and include methods which do notrequire extraction of lipid from the organism or part thereof, forexample, analysis by near infrared (NIR) or nuclear magnetic resonance(NMR).

In a further embodiment, the level of the one or more non-polar lipidsand/or the total non-polar lipid content of the vegetative plant part,or non-human organism or part thereof, or seed is at least 0.5% greateron a dry weight or seed weight basis and/or at least 1% greater on arelative basis, preferably at least 1% or 2% greater on a dry weight orseed weight basis, than a corresponding vegetative plant part, ornon-human organism or a part thereof, or seed lacking the one or moreexogenous polynucleotides but comprising an exogenous polynucleotideencoding an Arabidopsis thaliana DGAT1 (SEQ ID NO:83).

In yet a further embodiment, the vegetative plant part or the non-humanorganism or part thereof, or seed further comprises (i) one or moreintroduced mutations, and/or (ii) an exogenous polynucleotide whichdown-regulate the production and/or activity of an endogenous enzyme ofthe vegetative plant part or the non-human organism or part thereof, theendogenous enzyme being selected from a fatty acid acyltransferase suchas DGAT, sn-1 glycerol-3-phosphate acyltransferase (sn-1 GPAT),1-acyl-glycerol-3-phosphate acyltransferase (LPAAT),acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT), phosphatidicacid phosphatase (PAP), an enzyme involved in starch biosynthesis suchas (ADP)-glucose pyrophosphorylase (AGPase), a fatty acid desaturasesuch as a Δ12 fatty acid desaturase (FAD2), a polypeptide involved inthe degradation of lipid and/or which reduces lipid content such as alipase such as CGi58 polypeptide or SUGAR-DEPENDENT1 triacylglycerollipase, or a combination of two or more thereof. In an alternativeembodiment, the vegetative plant part or the non-human organism or partthereof does not comprise (i) above, or does not comprise (ii) above, ordoes not comprise (i) above and does not comprise (ii) above. In anembodiment, the exogenous polynucleotide which down-regulates theproduction of AGPase is not the polynucleotide disclosed in Sanjaya etal. (2011). In an embodiment, the exogenous polynucleotides in thevegetative plant part or the non-human organism or part thereof, or seeddoes not consist of an exogenous polynucleotide encoding a WRI1 and anexogenous polynucleotide encoding an RNA molecule which inhibitsexpression of a gene encoding an AGPase.

In the process of either the first or second aspects, the vegetativeplant part, or non-human organism or part thereof, or seed, or theextracted lipid or oil, is further defined in preferred embodiments.Therefore, in an embodiment one or more or all of the following featuresapply

(i) oleic acid comprises at least 20% (mol %), at least 22% (mol %), atleast 30% (mol %), at least 40% (mol %), at least 50% (mol %), or atleast 60% (mol %), preferably at least 65% (mol %) or at least 66% (mol%) of the total fatty acid content in the non-polar lipid or oil in thevegetative plant part, non-human organism or part thereof, or seed,

ii) oleic acid comprises at least 20% (mol %), at least 22% (mol %), atleast 30% (mol %), at least 40% (mol %), at least 50% (mol %), or atleast 60% (mol %), preferably at least 65% (mol %) or at least 66% (mol%) of the total fatty acid content in the extracted lipid or oil,

(iii) the non-polar lipid or oil in the vegetative plant part, non-humanorganism or part thereof, or seed comprises a fatty acid which comprisesa hydroxyl group, an epoxy group, a cyclopropane group, a doublecarbon-carbon bond, a triple carbon-carbon bond, conjugated doublebonds, a branched chain such as a methylated or hydroxylated branchedchain, or a combination of two or more thereof, or any of two, three,four, five or six of the aforementioned groups, bonds or branchedchains, and

(iv) the extracted lipid or oil comprises a fatty acid which comprises ahydroxyl group, an epoxy group, a cyclopropane group, a doublecarbon-carbon bond, a triple carbon-carbon bond, conjugated doublebonds, a branched chain such as a methylated or hydroxylated branchedchain, or a combination of two or more thereof, or any of two, three,four, five or six of the aforementioned groups, bonds or branchedchains. The fatty acid composition in this embodiment is measured priorto any modification of the fatty acid composition, such as, for example,by fractionating the extracted lipid or oil to alter the fatty acidcomposition. In preferred embodiments, the extent of the increase is asdefined in Feature (i).

In an embodiment, the level of a lipid in the vegetative plant part,non-human organism or part thereof, or seed and/or in the extractedlipid or oil is determinable by analysis by using gas chromatography offatty acid methyl esters prepared from the extracted lipid or oil. Themethod of analysis is preferably as described in Example 1 herein.

Again with respect to either the first or second aspects, the inventionprovides for one or more exogenous polynucleotides in the vegetativeplant part, or non-human organism or part thereof, or seed used in theprocess. Therefore, in an embodiment, the vegetative plant part, or thenon-human organism or part thereof, or the seed comprises a firstexogenous polynucleotide which encodes an RNA or preferably atranscription factor polypeptide that increases the expression of one ormore glycolytic or fatty acid biosynthetic genes in a vegetative plantpart, or a non-human organism or a part thereof, or a seed,respectively, and a second exogenous polynucleotide which encodes an RNAor a polypeptide involved in biosynthesis of one or more non-polarlipids, wherein the first and second exogenous polynucleotides are eachoperably linked to a promoter which is capable of directing expressionof the polynucleotide in a vegetative plant part, or a non-humanorganism or a part thereof, or a seed, respectively. That is, the firstand second exogenous polynucleotides encode different factors whichtogether provide for the increase in the non-polar lipid content in thevegetative plant part, or the non-human organism or part thereof, or theseed.

The increase is preferably additive, more preferably synergistic,relative to the presence of either the first or second exogenouspolynucleotide alone. The factors encoded by the first and secondpolynucleotides operate by different mechanisms. Preferably, thetranscription factor polypeptide increases the availability ofsubstrates for non-polar lipid synthesis, such as, for example,increasing glycerol-3-phosphate and/or fatty acids preferably in theform of acyl-CoA, by increasing expression of genes, for example atleast 5 or at least 8 genes, involved in glycolysis or fatty acidbiosynthesis (such as, but not limited to, one or more of ACCase,sucrose transporters (SuSy, cell wall invertases), ketoacyl synthase(KAS), phosphofructokinase (PFK), pyruvate kinase (PK) (for example,(At5g52920, At3g22960), pyruvate dehydrogenase, hexose transporters (forexample, GPT2 and PPT1), cytosolic fructokinase, cytosolicphosphoglycerate mutase, enoyl-ACP reductase (At2g05990), andphosphoglycerate mutase (At1g22170)) preferably more than one gene foreach category. In an embodiment, the first exogenous polynucleotideencodes a Wrinkled 1 (WRI1) transcription factor, a Leafy Cotyledon 1(Lec1) transcription factor, a Leafy Cotyledon 2 (LEC2) transcriptionfactor, a Fus3 transcription factor, an ABI3 transcription factor, aDof4 transcription factor, a BABY BOOM (BBM) transcription factor or aDof11 transcription factor. In one embodiment, the LEC2 is not anArabidopsis LEC2. As part of this embodiment, or separately, the secondexogenous polynucleotide may encode a polypeptide having a fatty acidacyltransferase activity, for example, monoacylglycerol acyltransferase(MGAT) activity and/or diacylglycerol acyltransferase (DGAT) activity,or glycerol-3-phosphate acyltransferase (GPAT) activity. In oneembodiment, the DGAT is not an Arabidopsis DGAT.

In a preferred embodiment, the vegetative plant part, or non-humanorganism or a part thereof, or the seed, of the first or second aspectsof the invention comprises two or more exogenous polynucleotide(s), oneof which encodes a transcription factor polypeptide that increases theexpression of one or more glycolytic or fatty acid biosynthetic genes inthe vegetative plant part, or non-human organism or a part thereof, orseed such as a Wrinkled 1 (WRI1) transcription factor, and a second ofwhich encodes a polypeptide involved in biosynthesis of one or morenon-polar lipids such as a DGAT.

In an embodiment, the vegetative plant part, non-human organism or apart thereof, or the seed of the first or second aspects of theinvention may further comprise a third, or more, exogenouspolynucleotide(s). The third, or more, exogenous polynucleotide(s) mayencode one or more or any combination of:

i) a further RNA or transcription factor polypeptide that increases theexpression of one or more glycolytic or fatty acid biosynthetic genes ina non-human organism or a part thereof (for example, if the firstexogenous polynucleotide encodes a Wrinkled 1 (WRI1) transcriptionfactor, the third exogenous polynucleotide may encode a LEC2 or BBMtranscription factor (preferably, LEC2 or BBM expression controlled byan inducible promoter or a promoter which does not result in hightransgene expression levels),

ii) a further RNA or polypeptide involved in biosynthesis of one or morenon-polar lipids (for example, if the second exogenous polynucleotideencodes a DGAT, the third exogenous polynucleotide may encode a MGAT orGPAT, or two further exogenous polynucleotides may be present encodingan MGAT and a GPAT),

iii) a polypeptide that stabilizes the one or more non-polar lipids,preferably an oleosin, such as a polyoleosin or a caleosin, morepreferably a polyoleosin,

iv) an RNA molecule which inhibits expression of a gene encoding apolypeptide involved in starch biosynthesis such as a AGPasepolypeptide,

v) an RNA molecule which inhibits expression of a gene encoding apolypeptide involved in the degradation of lipid and/or which reduceslipid content such as a lipase such as CGi58 polypeptide orSUGAR-DEPENDENT1 triacylglycerol lipase, or

vi) a silencing suppressor polypeptide,

wherein the third, or more, exogenous polynucleotide(s) is operablylinked to a promoter which is capable of directing expression of thepolynucleotide(s) in a vegetative plant part, or a non-human organism ora part thereof, or a seed, respectively.

A number of specific combinations of genes are shown herein to beeffective for increasing non-polar lipid contents. Therefore, regardingthe process of either the first or second aspects of the invention, inan embodiment, the vegetative plant part, or the non-human organism orpart thereof, or the seed comprises one or more exogenouspolynucleotide(s) which encode:

i) a Wrinkled 1 (WRI1) transcription factor and a DGAT,

ii) a WRI1 transcription factor and a DGAT and an Oleosin,

iii) a WRI1 transcription factor, a DGAT, a MGAT and an Oleosin,

iv) a monoacylglycerol acyltransferase (MGAT),

v) a diacylglycerol acyltransferase 2 (DGAT2),

vi) a MGAT and a glycerol-3-phosphate acyltransferase (GPAT),

vii) a MGAT and a DGAT,

viii) a MGAT, a GPAT and a DGAT,

ix) a WRI1 transcription factor and a MGAT,

x) a WRI1 transcription factor, a DGAT and a MGAT,

xi) a WRI1 transcription factor, a DGAT, a MGAT, an Oleosin and a GPAT,

xii) a DGAT and an Oleosin, or

xiii) a MGAT and an Oleosin, and

xiv) optionally, a silencing suppressor polypeptide,

wherein each of the one or more exogenous polynucleotide(s) is operablylinked to a promoter which is capable of directing expression of thepolynucleotide in a vegetative plant part, or a non-human organism orpart thereof, or seed, respectively. Preferably the one or moreexogenous polynucleotides are stably integrated into the genome of thevegetative plant part, or the non-human organism or part thereof, or theseed, and more preferably are present in a homozygous state. Thepolynucleotide may encode an enzyme having an amino acid sequence whichis the same as a sequence of a naturally occurring enzyme of, forexample, plant, yeast or animal origin. Further, the polynucleotide mayencode an enzyme having one or more conservative mutations when comparedto the naturally occurring enzyme.

In an embodiment,

(i) the GPAT also has phosphatase activity to produce MAG, such as apolypeptide having an amino acid sequence of Arabidopsis GPAT4 or GPAT6,and/or

(ii) the DGAT is a DGAT1 or a DGAT2, and/or

(iii) the MGAT is an MGAT1 or an MGAT2.

In a preferred embodiment, the vegetative plant part, the non-humanorganism or part thereof, or the seed comprises a first exogenouspolynucleotide encoding a WRI1 and a second exogenous polynucleotideencoding a DGAT, preferably a DGAT1.

In another preferred embodiment, the vegetative plant part, thenon-human organism or part thereof, or the seed comprises a firstexogenous polynucleotide encoding a WRI1, a second exogenouspolynucleotide encoding a DGAT, preferably a DGAT1, and a thirdexogenous polynucleotide encoding an oleosin.

In a further embodiment, the vegetative plant part, the non-humanorganism or part thereof, or the seed comprises a first exogenouspolynucleotide encoding a WRI1, a second exogenous polynucleotideencoding a DGAT, preferably a DGAT1, a third exogenous polynucleotideencoding an oleosin, and a fourth exogenous polynucleotide encoding anMGAT, preferably an MGAT2.

In a further embodiment, the vegetative plant part, the non-humanorganism or part thereof, or the seed comprises a first exogenouspolynucleotide encoding a WRI1, a second exogenous polynucleotideencoding a DGAT, preferably a DGAT1, a third exogenous polynucleotideencoding an oleosin, and a fourth exogenous polynucleotide encoding LEC2or BBM.

In a further embodiment, the vegetative plant part, the non-humanorganism or part thereof, or the seed comprises a first exogenouspolynucleotide encoding a WRI1, a second exogenous polynucleotideencoding a DGAT, preferably a DGAT1, a third exogenous polynucleotideencoding an oleosin, a fourth exogenous polynucleotide encoding an MGAT,preferably an MGAT2, and a fifth exogenous polynucleotide encoding LEC2or BBM.

In a further embodiment, the vegetative plant part, the non-humanorganism or part thereof, or the seed comprises a first exogenouspolynucleotide encoding a WRI1, a second exogenous polynucleotideencoding a DGAT, preferably a DGAT1, a third exogenous polynucleotideencoding an oleosin, and a fourth exogenous polynucleotide encoding anRNA molecule which inhibits expression of a gene encoding a lipase suchas CGi58 polypeptide.

In a further embodiment, the vegetative plant part, the non-humanorganism or part thereof, or the seed comprises a first exogenouspolynucleotide encoding a WRI1, a second exogenous polynucleotideencoding a DGAT, preferably a DGAT1, a third exogenous polynucleotideencoding an oleosin, a fourth exogenous polynucleotide encoding an RNAmolecule which inhibits expression of a gene encoding a lipase such as aCGi58 polypeptide, and a fifth exogenous polynucleotide encoding LEC2 orBBM.

In a further embodiment, the vegetative plant part, the non-humanorganism or part thereof, or the seed comprises a first exogenouspolynucleotide encoding a WRI1, a second exogenous polynucleotideencoding a DGAT, preferably a DGAT1, a third exogenous polynucleotideencoding an oleosin, a fourth exogenous polynucleotide encoding an RNAmolecule which inhibits expression of a gene encoding a lipase such as aCGi58 polypeptide, and a fifth exogenous polynucleotide encoding anMGAT, preferably an MGAT2.

In a further embodiment, the vegetative plant part, the non-humanorganism or part thereof, or the seed comprises a first exogenouspolynucleotide encoding a WRI1, a second exogenous polynucleotideencoding a DGAT, preferably a DGAT1, a third exogenous polynucleotideencoding an oleosin, a fourth exogenous polynucleotide encoding an RNAmolecule which inhibits expression of a gene encoding a lipase such as aCGi58 polypeptide, a fifth exogenous polynucleotide encoding an MGAT,preferably an MGAT2, and a sixth exogenous polynucleotide encoding LEC2or BBM.

In an embodiment, the seed comprises a first exogenous polynucleotideencoding a WRI1, a second exogenous polynucleotide encoding a DGAT,preferably a DGAT1, a third exogenous polynucleotide encoding anoleosin, and a fourth exogenous polynucleotide encoding an MGAT,preferably an MGAT2. Preferably, the seed further comprises a fifthexogenous polynucleotide encoding a GPAT.

Where relevant, instead of a polynucleotide encoding an RNA moleculewhich inhibits expression of a gene encoding a lipase such as a CGi58polypeptide, the vegetative plant part, the non-human organism or partthereof, or the seed has one or more introduced mutations in the lipasegene such as a CGi58 gene which confers reduced levels of the lipasepolypeptide when compared to a corresponding vegetative plant part,non-human organism or part thereof, or seed lacking the mutation.

In a preferred embodiment, the exogenous polynucleotides encoding theDGAT and oleosin are operably linked to a constitutive promoter, or apromoter active in green tissues of a plant at least before and up untilflowering, which is capable of directing expression of thepolynucleotides in the vegetative plant part, the non-human organism orpart thereof, or the seed. In a further preferred embodiment, theexogenous polynucleotide encoding WRI1, and RNA molecule which inhibitsexpression of a gene encoding a lipase such as a CGi58 polypeptide, isoperably linked to a constitutive promoter, a promoter active in greentissues of a plant at least before and up until flowering, or aninducible promoter, which is capable of directing expression of thepolynucleotides in the vegetative plant part, the non-human organism orpart thereof, or the seed. In yet a further preferred embodiment, theexogenous polynucleotides encoding LEC2, BBM and/or MGAT2 are operablylinked to an inducible promoter which is capable of directing expressionof the polynucleotides in the vegetative plant part, the non-humanorganism or part thereof, or the seed.

In each of the above embodiments, the polynucleotides may be provided asseparate molecules or may be provided as a contiguous single molecule,such as on a single T-DNA molecule. In an embodiment, the orientation oftranscription of at least one gene on the T-DNA molecule is opposite tothe orientation of transcription of at least one other gene on the T-DNAmolecule.

In each of the above embodiments, the total non-polar lipid content ofthe vegetative plant part, or non-human organism or part thereof, or theseed, preferably a plant leaf or part thereof, stem or tuber, is atleast about 3%, more preferably at least about 5%, preferably at leastabout 7%, more preferably at least about 10%, more preferably at leastabout 11%, more preferably at least about 12%, more preferably at leastabout 13%, more preferably at least about 14%, or more preferably atleast about 15% (w/w dry weight). In a further preferred embodiment, thetotal non-polar lipid content is between 5% and 25%, between 7% and 25%,between 10% and 25%, between 12% and 25%, between 15% and 25%, between7% and 20%, between 10% and 20%, between 10% and 15%, between 15% and20%, between 20% and 25%, about 10%, about 11%, about 12%, about 13%,about 14%, about 15%, about 16%, about 17%, about 18%, about 20%, orabout 22%, each as a percentage of dry weight or seed weight. In aparticularly preferred embodiment, the vegetative plant part is a leaf(or leaves) or a portion thereof. In a more preferred embodiment, thevegetative plant part is a leaf portion having a surface area of atleast 1 cm².

Furthermore, in each of the above embodiments, the total TAG content ofthe vegetative plant part, or non-human organism or part thereof, or theseed, preferably a plant leaf or part thereof, stem or tuber, is atleast about 3%, more preferably at least about 5%, preferably at leastabout 7%, more preferably at least about 10%, more preferably at leastabout 11%, more preferably at least about 12%, more preferably at leastabout 13%, more preferably at least about 14%, more preferably at leastabout 15%, or more preferably at least about 17% (w/w dry weight). In afurther preferred embodiment, the total TAG content is between 5% and30%, between 7% and 30%, between 10% and 30%, between 12% and 30%,between 15% and 30%, between 7% and 30%, between 10% and 30%, between20% and 28%, between 18% and 25%, between 22% and 30%, about 10%, about11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%,about 18%, about 20%, or about 22%, each as a percentage of dry weightor seed weight. In a particularly preferred embodiment, the vegetativeplant part is a leaf (or leaves) or a portion thereof. In a morepreferred embodiment, the vegetative plant part is a leaf portion havinga surface area of at least 1 cm².

Furthermore, in each of the above embodiments, the total lipid contentof the vegetative plant part, or non-human organism or part thereof, orthe seed, preferably a plant leaf or part thereof, stem or tuber, is atleast about 3%, more preferably at least about 5%, preferably at leastabout 7%, more preferably at least about 10%, more preferably at leastabout 11%, more preferably at least about 12%, more preferably at leastabout 13%, more preferably at least about 14%, more preferably at leastabout 15%, more preferably at least about 17% (w/w dry weight), morepreferably at least about 20%, more preferably at least about 25%. In afurther preferred embodiment, the total lipid content is between 5% and35%, between 7% and 35%, between 10% and 35%, between 12% and 35%,between 15% and 35%, between 7% and 35%, between 10% and 20%, between18% and 28%, between 20% and 28%, between 22% and 28%, about 10%, about11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%,about 18%, about 20%, about 22%, or about 25%, each as a percentage ofdry weight. Typically, the total lipid content of the vegetative plantpart, or non-human organism or part thereof is about 2-3% higher thanthe non-polar lipid content. In a particularly preferred embodiment, thevegetative plant part is a leaf (or leaves) or a portion thereof. In amore preferred embodiment, the vegetative plant part is a leaf portionhaving a surface area of at least 1 cm².

In an embodiment, the vegetative plant part, the non-human organism orpart thereof, or the seed, preferably the vegetative plant part,comprises a first exogenous polynucleotide encoding a WRI1, a secondexogenous polynucleotide encoding a DGAT, preferably a DGAT1, a thirdexogenous polynucleotide encoding an MGAT, preferably an MGAT2, and afourth exogenous polynucleotide encoding an oleosin, wherein thevegetative plant part, non-human organism or part thereof, or seed hasone or more or all of the following features:

i) a total lipid content of at least 8%, at least 10%, at least 12%, atleast 14%, or at least 15.5% (% weight),

ii) at least a 3 fold, at least a 5 fold, at least a 7 fold, at least an8 fold, or least a 10 fold, at higher total lipid content in thevegetative plant part or non-human organism relative to a correspondingvegetative plant part or non-human organism lacking the exogenouspolynucleotides,

iii) a total TAG content of at least 5%, at least 6%, at least 6.5% orat least 7% (% weight of dry weight or seed weight),

iv) at least a 40 fold, at least a 50 fold, at least a 60 fold, or atleast a 70 fold, or at least a 100 fold, higher total TAG contentrelative to a corresponding vegetative plant part or non-human organismlacking the exogenous polynucleotides,

v) oleic acid comprises at least 15%, at least 19% or at least 22% (%weight) of the fatty acids in TAG,

vi) at least a 10 fold, at least a 15 fold or at least a 17 fold higherlevel of oleic acid in TAG relative to a corresponding vegetative plantpart or non-human organism lacking the exogenous polynucleotides,

vii) palmitic acid comprises at least 20%, at least 25%, at least 30% orat least 33% (% weight) of the fatty acids in TAG,

viii) at least a 1.5 fold higher level of palmitic acid in TAG relativeto a corresponding vegetative plant part or non-human organism lackingthe exogenous polynucleotides,

ix) linoleic acid comprises at least 22%, at least 25%, at least 30% orat least 34% (% weight) of the fatty acids in TAG,

x) α-linolenic acid comprises less than 20%, less than 15%, less than11% or less than 8% (% weight) of the fatty acids in TAG, and

xi) at least a 5 fold, or at least an 8 fold, lower level of α-linolenicacid in TAG relative to a corresponding vegetative plant part or nonhuman organism lacking the exogenous polynucleotides. In thisembodiment, preferably the vegetative plant part at least hasfeature(s), i), ii) iii), iv), i) and ii), i) and iii), i) and iv), i)to iii), i), iii) and iv), i) to iv), ii) and iii), ii) and iv), ii) toiv), or iii) and iv). In an embodiment, % dry weight is % leaf dryweight.

In a further embodiment, the vegetative plant part, the non-humanorganism or part thereof, or the seed, preferably the vegetative plantpart, comprises a first exogenous polynucleotide encoding a WRI1, asecond exogenous polynucleotide encoding a DGAT, preferably a DGAT1, athird exogenous polynucleotide encoding an oleosin, wherein thevegetative plant part, non-human organism or part thereof, or seed hasone or more or all of the following features:

i) a total TAG content of at least 10%, at least 12.5%, at least 15% orat least 17% (% weight of dry weight or seed weight),

ii) at least a 40 fold, at least a 50 fold, at least a 60 fold, or atleast a 70 fold, or at least a 100 fold, higher total TAG content in thevegetative plant part or non-human organism relative to a correspondingvegetative plant part or non human organism lacking the exogenouspolynucleotides,

iii) oleic acid comprises at least 19%, at least 22%, or at least 25% (%weight) of the fatty acids in TAG,

iv) at least a 10 fold, at least a 15 fold, at least a 17 fold, or atleast a 19 fold, higher level of oleic acid in TAG in the vegetativeplant part or non-human organism relative to a corresponding vegetativeplant part or non-human organism lacking the exogenous polynucleotides,

v) palmitic acid comprises at least 20%, at least 25%, or at least 28%(% weight) of the fatty acids in TAG,

vi) at least a 1.25 fold higher level of palmitic acid in TAG in thevegetative plant part or non-human organism relative to a correspondingvegetative plant part or non-human organism lacking the exogenouspolynucleotides,

vii) linoleic acid comprises at least 15%, or at least 20%, (% weight)of the fatty acids in TAG,

viii) α-linolenic acid comprises less than 15%, less than 11% or lessthan 8% (% weight) of the fatty acids in TAG, and

ix) at least a 5 fold, or at least an 8 fold, lower level of α-linolenicacid in TAG in the vegetative plant part or non-human organism relativeto a corresponding vegetative plant part or non-human organism lackingthe exogenous polynucleotides. In this embodiment, preferably thevegetative plant part at least has feature(s), i), ii), or i) and ii).In an embodiment, % dry weight is % leaf dry weight.

Preferably, the defined features for the two above embodiments are as atthe flowering stage of the plant.

In an alternate embodiment, the vegetative plant part, the non-humanorganism or part thereof, or the seed consists of one or more exogenouspolynucleotides encoding a DGAT1 and a LEC2.

In a preferred embodiment, the exogenous polynucleotide encoding WRI1comprises one or more of the following:

i) nucleotides whose sequence is set forth as any one of SEQ ID NOs:231to 278,

ii) nucleotides encoding a polypeptide comprising amino acids whosesequence is set forth as any one of SEQ ID NOs:279 to 337, or abiologically active fragment thereof,

iii) nucleotides whose sequence is at least 30% identical to i) or ii),and

iv) nucleotides which hybridize to any one of i) to iii) under stringentconditions.

In a preferred embodiment, the exogenous polynucleotide encoding DGATcomprises one or more of the following:

i) nucleotides whose sequence is set forth as any one of SEQ ID NOs:204to 211, 338 to 346,

ii) nucleotides encoding a polypeptide comprising amino acids whosesequence is set forth as any one of SEQ ID NOs:83, 212 to 219, 347 to355, or a biologically active fragment thereof,

iii) nucleotides whose sequence is at least 30% identical to i) or ii),and

iv) a polynucleotide which hybridizes to any one of i) to iii) understringent conditions.

In another preferred embodiment, the exogenous polynucleotide encodingMGAT comprises one or more of the following:

i) nucleotides whose sequence is set forth as any one of SEQ ID NOs:1 to44,

ii) nucleotides encoding a polypeptide comprising amino acids whosesequence is set forth as any one of SEQ ID NOs:45 to 82, or abiologically active fragment thereof,

iii) nucleotides whose sequence is at least 30% identical to i) or ii),and

iv) a polynucleotide which hybridizes to any one of i) to iii) understringent conditions.

In another preferred embodiment, the exogenous polynucleotide encodingGPAT comprises one or more of the following:

i) nucleotides whose sequence is set forth as any one of SEQ ID NOs:84to 143,

ii) nucleotides encoding a polypeptide comprising amino acids whosesequence is set forth as any one of SEQ ID NOs:144 to 203, or abiologically active fragment thereof,

iii) nucleotides whose sequence is at least 30% identical to i) or ii),and

iv) a polynucleotide which hybridizes to any one of i) to iii) understringent conditions.

In another preferred embodiment, the exogenous polynucleotide encodingDGAT2 comprises one or more of the following:

i) nucleotides whose sequence is set forth as any one of SEQ ID NOs:204to 211,

ii) nucleotides encoding a polypeptide comprising amino acids whosesequence is set forth as any one of SEQ ID NOs:212 to 219, or abiologically active fragment thereof,

iii) nucleotides whose sequence is at least 30% identical to i) or ii),and

iv) a polynucleotide which hybridizes to any one of i) to iii) understringent conditions.

In another preferred embodiment, the exogenous polynucleotide encodingan oleosin comprises one or more of the following:

i) nucleotides whose sequence is set forth as any one of SEQ ID NOs:389to 408,

ii) nucleotides encoding a polypeptide comprising amino acids whosesequence is set forth as any one of SEQ ID NOs:362 to 388, or abiologically active fragment thereof,

iii) nucleotides whose sequence is at least 30% identical to i) or ii),and

iv) a sequence of nucleotides which hybridizes to any one of i) to iii)under stringent conditions.

In an embodiment, the CGi58 polypeptide comprises one or more of thefollowing:

i) nucleotides whose sequence is set forth as any one of SEQ ID NOs:422to 428,

ii) nucleotides encoding a polypeptide comprising amino acids whosesequence is set forth as any one of SEQ ID NOs:429 to 436, or abiologically active fragment thereof,

iii) nucleotides whose sequence is at least 30% identical to i) or ii),and

iv) a sequence of nucleotides which hybridizes to any one of i) to iii)under stringent conditions.

In another embodiment, the exogenous polynucleotide encoding LEC2comprises one or more of the following:

i) nucleotides whose sequence is set forth as any one of SEQ ID NOs:437to 439,

ii) nucleotides encoding a polypeptide comprising amino acids whosesequence is set forth as any one of SEQ ID NOs:442 to 444, or abiologically active fragment thereof,

iii) nucleotides whose sequence is at least 30% identical to i) or ii),and

iv) a sequence of nucleotides which hybridizes to any one of i) to iii)under stringent conditions.

In a further embodiment, the exogenous polynucleotide encoding BBMcomprises one or more of the following:

i) nucleotides whose sequence is set forth as any one of SEQ ID NOs:440or 441

ii) nucleotides encoding a polypeptide comprising amino acids whosesequence is set forth as any one of SEQ ID NOs:445 or 446, or abiologically active fragment thereof,

iii) nucleotides whose sequence is at least 30% identical to i) or ii),and

iv) a sequence of nucleotides which hybridizes to any one of i) to iii)under stringent conditions.

Clearly, sequences preferred in one embodiment can be combined withsequences preferred in another embodiment and more advantageouslyfurther combined with a sequence preferred in yet another embodiment.

In one embodiment, the one or more exogenous polynucleotides encode amutant MGAT and/or DGAT and/or GPAT. For example, the one or moreexogenous polynucleotides may encode a MGAT and/or DGAT and/or GPAThaving one, or more than one, conservative amino acid substitutions asexemplified in Table 1 relative to a wildtype MGAT and/or DGAT and/orGPAT as defined by a SEQ ID NO herein. Preferably the mutant polypeptidehas an equivalent or greater activity relative to the non-mutantpolypeptide.

In an embodiment, the vegetative plant part, non-human organism or partthereof, or seed comprises a first exogenous polynucleotide that encodesa MGAT and a second exogenous polynucleotide that encodes a GPAT. Thefirst and second polynucleotides may be provided as separate moleculesor may be provided as a contiguous single molecule, such as on a singleT-DNA molecule. In an embodiment, the orientation of transcription of atleast one gene on the T-DNA molecule is opposite to the orientation oftranscription of at least one other gene on the T-DNA molecule. In apreferred embodiment, the GPAT is a GPAT having phosphatase activitysuch as an Arabidopsis GPAT4 or GPAT6. The GPAT having phosphataseactivity acts to catalyze the formation of MAG from G-3-P (i.e.,acylates G-3-P to form LPA and subsequently removes a phosphate group toform MAG) in the non-human organism or part thereof. The MGAT then actsto catalyze the formation of DAG in the non-human organism or partthereof by acylating the MAG with an acyl group derived from fattyacyl-CoA. The MGAT such as A. thaliana MGAT1 may also act to catalyzethe formation of TAG in the non-human organism or part thereof if italso has DGAT activity.

The vegetative plant part, non-human organism or part thereof, or seedmay comprise a third exogenous polynucleotide encoding, for example, aDGAT. The first, second and third polynucleotides may be provided asseparate molecules or may be provided as a contiguous single molecule,such as on a single T-DNA molecule. The DGAT acts to catalyse theformation of TAG in the transgenic vegetative plant part, non-humanorganism or part thereof, or seed by acylating the DAG (preferablyproduced by the MGAT pathway) with an acyl group derived from fattyacyl-CoA. In an embodiment, the orientation of transcription of at leastone gene on the T-DNA molecule is opposite to the orientation oftranscription of at least one other gene on the T-DNA molecule.

In another embodiment, the vegetative plant part, non-human organism orpart thereof, or seed comprises a first exogenous polynucleotide thatencodes a MGAT and a second exogenous polynucleotide that encodes aDGAT. The first and second polynucleotides may be provided as separatemolecules or may be provided as a contiguous single molecule, such as ona single T-DNA molecule. In an embodiment, the orientation oftranscription of at least one gene on the T-DNA molecule is opposite tothe orientation of transcription of at least one other gene on the T-DNAmolecule. The vegetative plant part, non-human organism or part thereof,or seed may comprise a third exogenous polynucleotide encoding, forexample, a GPAT, preferably a GPAT having phosphatase activity such asan Arabidopsis GPAT4 or GPAT6. The first, second and thirdpolynucleotides may be provided as separate molecules or may be providedas a contiguous single molecule.

Furthermore, an endogenous gene activity in the plant, vegetative plantpart, or the non-human organism or part thereof, or the seed may bedown-regulated. Therefore, in an embodiment, the vegetative plant part,the non-human organism or part thereof, or the seed comprises one ormore of:

(i) one or more introduced mutations in a gene which encodes anendogenous enzyme of the plant, vegetative plant part, non-humanorganism or part thereof, or seed, respectively, or

(ii) an exogenous polynucleotide which down-regulates the productionand/or activity of an endogenous enzyme of the plant, vegetative plantpart, non-human organism or part thereof, or seed, respectively,

wherein each endogenous enzyme is selected from the group consisting ofa fatty acid acyltransferase such as DGAT, an sn-1 glycerol-3-phosphateacyltransferase (sn-1 GPAT), a 1-acyl-glycerol-3-phosphateacyltransferase (LPAAT), an acyl-CoA:lysophosphatidylcholineacyltransferase (LPCAT), a phosphatidic acid phosphatase (PAP), anenzyme involved in starch biosynthesis such as (ADP)-glucosepyrophosphorylase (AGPase), a fatty acid desaturase such as a Δ12 fattyacid desaturase (FAD2), a polypeptide involved in the degradation oflipid and/or which reduces lipid content such as a lipase such as aCGi58 polypeptide or SUGAR-DEPENDENT1 triacylglycerol lipase, or acombination of two or more thereof. In an embodiment, the exogenouspolynucleotide is selected from the group consisting of an antisensepolynucleotide, a sense polynucleotide, a catalytic polynucleotide, amicroRNA, a polynucleotide which encodes a polypeptide which binds theendogenous enzyme, a double stranded RNA molecule or a processed RNAmolecule derived therefrom. In an embodiment, the exogenouspolynucleotide which down-regulates the production of AGPase is not thepolynucleotide disclosed in Sanjaya et al. (2011). In an embodiment, theexogenous polynucleotides in the vegetative plant part or the non-humanorganism or part thereof, or seed does not consist of an exogenouspolynucleotide encoding a WRI1 and an exogenous polynucleotide encodingan RNA molecule which inhibits expression of a gene encoding an AGPase.

Increasing the level of non-polar lipids is important for applicationsinvolving particular fatty acids. Therefore, in an embodiment, the totalnon-polar lipid, the extracted lipid or oil comprises:

(i) non-polar lipid which is TAG, DAG, TAG and DAG, or MAG, and

(ii) a specific PUFA which is EDA, ARA, SDA, ETE, ETA, EPA, DPA, DHA,the specific PUFA being at a level of at least 1% of the total fattyacid content in the non-polar lipid, or a combination of two or more ofthe specific PUFA, or

(iii) a fatty acid which is present at a level of at least 1% of thetotal fatty acid content in the non-polar lipid and which comprises ahydroxyl group, an epoxy group, a cyclopropane group, a doublecarbon-carbon bond, a triple carbon-carbon bond, conjugated doublebonds, a branched chain such as a methylated or hydroxylated branchedchain, or a combination of two or more thereof, or any of two, three,four, five or six of the aforementioned groups, bonds or branchedchains.

In a third aspect, the invention provides non-human organisms,preferably plants, or parts thereof such as vegetative plant parts orseed, which are useful in the processes of the first and second aspectsor in further aspects described hereafter. Each of the features in theembodiments described for the first and second aspects can be appliedmutatis mutandis to the non-human organisms, preferably plants, or partsthereof such as vegetative plant parts or seed of the third aspect.Particular embodiments are emphasized as follows.

In an embodiment of the third aspect, the present invention provides anon-human organism or a part thereof, wherein the non-human organism orpart thereof has a total non-polar lipid content of at least about 3%,more preferably at least about 5%, preferably at least about 7%, morepreferably at least about 10%, more preferably at least about 11%, morepreferably at least about 12%, more preferably at least about 13%, morepreferably at least about 14%, or more preferably at least about 15%(w/w dry weight or seed weight), wherein one or more or all of thefollowing features apply:

(a) the non-human organism or a part thereof comprises one or moreexogenous polynucleotide(s) and an increased level of one or morenon-polar lipid(s) relative to a corresponding non-human organism or apart thereof, respectively, lacking the one or more exogenouspolynucleotide(s), wherein each of the one or more exogenouspolynucleotides is operably linked to a promoter which is capable ofdirecting expression of the polynucleotide in a non-human organism orpart thereof,

(b) the non-human organism is an alga selected from the group consistingof diatoms (bacillariophytes), green algae (chlorophytes), blue-greenalgae (cyanophytes), golden-brown algae (chrysophytes), haptophytes,brown algae and heterokont algae,

(c) the one or more non-polar lipid(s) comprise a fatty acid whichcomprises a hydroxyl group, an epoxy group, a cyclopropane group, adouble carbon-carbon bond, a triple carbon-carbon bond, conjugateddouble bonds, a branched chain such as a methylated or hydroxylatedbranched chain, or a combination of two or more thereof, or any of two,three, four, five or six of the aforementioned groups, bonds or branchedchains,

(d) the total fatty acid content in the non-polar lipid(s) comprises atleast 2% more oleic acid and/or at least 2% less palmitic acid than thenon-polar lipid(s) in the corresponding non-human organism or partthereof lacking the one or more exogenous polynucleotides of part (a),

(e) the non-polar lipid(s) comprise a modified level of total sterols,preferably free (non-esterified) sterols, steroyl esters, steroylglycosides, relative to the non-polar lipid(s) in the correspondingnon-human organism or part thereof lacking the one or more exogenouspolynucleotides of part (a),

(f) the non-polar lipid(s) comprise waxes and/or wax esters,

(g) the non-human organism or part thereof is one member of a pooledpopulation or collection of at least about 1000 such non-human organismsor parts thereof, respectively, from which the lipid is extracted.

In an embodiment of the third aspect, the invention provides a plantcomprising a vegetative part, or the vegetative part thereof, whereinthe vegetative part has a total non-polar lipid content of at leastabout 3%, more preferably at least about 5%, preferably at least about7%, more preferably at least about 10%, more preferably at least about11%, more preferably at least about 12%, more preferably at least about13%, more preferably at least about 14%, or more preferably at leastabout 15% (w/w dry weight). In a further preferred embodiment, the totalnon-polar lipid content is between 5% and 25%, between 7% and 25%,between 10% and 25%, between 12% and 25%, between 15% and 25%, between7% and 20%, between 10% and 20%, about 10%, about 11%, about 12%, about13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 20%,or about 22%, each as a percentage of dry weight. In a particularlypreferred embodiment, the vegetative plant part is a leaf (or leaves) ora portion thereof. In a more preferred embodiment, the vegetative plantpart is a leaf portion having a surface area of at least 1 cm². In afurther embodiment, the non-polar lipid comprises at least 90%triacylglycerols (TAG). Preferably the plant is fertile, morphologicallynormal, and/or agronomically useful. Seed of the plant preferablygerminates at a rate substantially the same as for a correspondingwild-type plant. Preferably the vegetative part is a leaf or a stem, ora combination of the two, or a root or tuber such as, for example,potato tubers.

In another embodiment, the non-human organism, preferably plant, or partthereof such as vegetative plant part or seed comprises one or moreexogenous polynucleotides as defined herein and has an increased levelof the one or more non-polar lipids and/or the total non-polar lipidcontent which is at least 2-fold, at least 3-fold, at least 4-fold, atleast 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, atleast 9-fold, at least 10-fold, or at least 12-fold, preferably at leastabout 13-fold or at least about 15-fold greater on a relative basis thana corresponding non-human organism, preferably plant, or part thereofsuch as vegetative plant part or seed lacking the one or more exogenouspolynucleotides.

In an embodiment, the invention provides a canola plant comprisingcanola seed whose oil content is at least 45% on a weight basis.Preferably, the canola plant or its seed have features as described inthe first and second aspects of the invention.

In an embodiment, the invention provides a corn plant comprising cornseed whose oil content is at least 5% on a weight basis. Preferably, thecorn plant or its seed have features as described in the first andsecond aspects of the invention.

In an embodiment, the invention provides a soybean plant comprisingsoybean seed whose oil content is at least 20% on a weight basis.Preferably, the soybean plant or its seed have features as described inthe first and second aspects of the invention.

In an embodiment, the invention provides a lupin plant comprising lupinseed whose oil content is at least 10% on a weight basis. Preferably,the lupin plant or its seed have features as described in the first andsecond aspects of the invention.

In an embodiment, the invention provides a peanut plant comprisingpeanuts whose oil content is at least 50% on a weight basis. Preferably,the peanut plant or its seed have features as described in the first andsecond aspects of the invention.

In an embodiment, the invention provides a sunflower plant comprisingsunflower seed whose oil content is at least 50% on a weight basis.Preferably, the sunflower plant or its seed have features as describedin the first and second aspects of the invention.

In an embodiment, the invention provides a cotton plant comprisingcotton seed whose oil content is at least 41% on a weight basis.Preferably, the cotton plant or its seed have features as described inthe first and second aspects of the invention.

In an embodiment, the invention provides a safflower plant comprisingsafflower seed whose oil content is at least 35% on a weight basis.Preferably, the safflower plant or its seed have features as describedin the first and second aspects of the invention.

In an embodiment, the invention provides a flax plant comprising flaxseed whose oil content is at least 36% on a weight basis. Preferably,the flax plant or its seed have features as described in the first andsecond aspects of the invention.

In an embodiment, the invention provides a Camelina sativa plantcomprising Camelina sativa seed whose oil content is at least 36% on aweight basis. Preferably, the Camelina sativa plant or its seed havefeatures as described in the first and second aspects of the invention.

In embodiments, the plants may be further defined by Features (i), (ii)and (iii) as described hereinbefore. In a preferred embodiment, theplant or the vegetative part comprises one or more or all of thefollowing features:

(i) oleic acid in a vegetative part or seed of the plant, the oleic acidbeing in an esterified or non-esterified form, wherein at least 20% (mol%), at least 22% (mol %), at least 30% (mol %), at least 40% (mol %), atleast 50% (mol %), or at least 60% (mol %), preferably at least 65% (mol%) or at least 66% (mol %) of the total fatty acids in the lipid contentof the vegetative part or seed is oleic acid,

(ii) oleic acid in a vegetative part or seed of the plant, the oleicacid being in an esterified form in non-polar lipid, wherein at least20% (mol %), at least 22% (mol %), at least 30% (mol %), at least 40%(mol %), at least 50% (mol %), or at least 60% (mol %), preferably atleast 65% (mol %) or at least 66% (mol %) of the total fatty acids inthe non-polar lipid content of the vegetative part or seed is oleicacid,

(iii) a modified fatty acid in a vegetative part or seed of the plant,the modified fatty acid being in an esterified or non-esterified form,preferably in an esterified form in non-polar lipids of the vegetativepart or seed, wherein the modified fatty acid comprises a hydroxylgroup, an epoxy group, a cyclopropane group, a double carbon-carbonbond, a triple carbon-carbon bond, conjugated double bonds, a branchedchain such as a methylated or hydroxylated branched chain, or acombination of two or more thereof, or any of two, three, four, five orsix of the aforementioned groups, bonds or branched chains, and

(iv) waxes and/or wax esters in the non-polar lipid of the vegetativepart or seed of the plant.

In an embodiment, the plant or the vegetative plant part is a member ofa population or collection of at least about 1000 such plants or parts.That is, each plant or plant part in the population or collection hasessentially the same properties or comprise the same exogenous nucleicacids as the other members of the population or collection. Preferably,the plants are homozygous for the exogenous polynucleotides, whichprovides a degree of uniformity. Preferably, the plants are growing in afield. The collection of vegetative plants parts have preferably beenharvested from plants growing in a field. Preferably, the vegetativeplant parts have been harvested at a time when the yield of non-polarlipids are at their highest. In one embodiment, the vegetative plantparts have been harvested about at the time of flowering. In anotherembodiment, the vegetative plant parts are harvested when the plants areat least about 1 month of age. In another embodiment, the vegetativeplant parts are harvested from about at the time of flowering to aboutthe beginning of senescence. In another embodiment, the vegetative plantparts are harvested at least about 1 month after induction of expressionof inducible genes.

In a further embodiment of the third aspect, the invention provides avegetative plant part, non-human organism or a part thereof, or seedcomprising one or more exogenous polynucleotide(s) and an increasedlevel of one or more non-polar lipid(s) relative to a correspondingvegetative plant part, non-human organism or a part thereof, or seedlacking the one or more exogenous polynucleotide(s), wherein each of theone or more exogenous polynucleotides is operably linked to a promoterwhich is capable of directing expression of the polynucleotide in avegetative plant part, non-human organism or part thereof, or seed andwherein one or more or all of the following features apply:

(i) the one or more exogenous polynucleotide(s) comprise a firstexogenous polynucleotide which encodes an RNA or transcription factorpolypeptide that increases the expression of one or more glycolytic orfatty acid biosynthetic genes in a vegetative plant part, non-humanorganism or a part thereof, or seed and a second exogenouspolynucleotide which encodes an RNA or polypeptide involved inbiosynthesis of one or more non-polar lipids,

(ii) if the non-human organism is a plant, a vegetative part of theplant has a total non-polar lipid content of at least about 3%, morepreferably at least about 5%, preferably at least about 7%, morepreferably at least about 10%, more preferably at least about 11%, morepreferably at least about 12%, more preferably at least about 13%, morepreferably at least about 14%, or more preferably at least about 15%(w/w dry weight),

(iii) the non-human organism is an alga selected from the groupconsisting of diatoms (bacillariophytes), green algae (chlorophytes),blue-green algae (cyanophytes), golden-brown algae (chrysophytes),haptophytes, brown algae and heterokont algae,

(iv) the non-polar lipid(s) comprise a fatty acid which comprises ahydroxyl group, an epoxy group, a cyclopropane group, a doublecarbon-carbon bond, a triple carbon-carbon bond, conjugated doublebonds, a branched chain such as a methylated or hydroxylated branchedchain, or a combination of two or more thereof, or any of two, three,four, five or six of the aforementioned groups, bonds or branchedchains,

(v) the vegetative plant part, non-human organism or part thereof, orseed comprises oleic acid in an esterified or non-esterified form in itslipid, wherein at least 20% (mol %), at least 22% (mol %), at least 30%(mol %), at least 40% (mol %), at least 50% (mol %), or at least 60%(mol %), preferably at least 65% (mol %) or at least 66% (mol %) of thetotal fatty acids in the lipid of the vegetative plant part, non-humanorganism or part thereof, or seed is oleic acid,

(vi) the vegetative plant part, non-human organism or part thereof, orseed comprises oleic acid in an esterified form in its non-polar lipid,wherein at least 20% (mol %), at least 22% (mol %), at least 30% (mol%), at least 40% (mol %), at least 50% (mol %), or at least 60% (mol %),preferably at least 65% (mol %) or at least 66% (mol %) of the totalfatty acids in the non-polar lipid of the vegetative plant part,non-human organism or part thereof, or seed is oleic acid,

(vii) the total fatty acid content in the lipid of the vegetative plantpart, non-human organism or part thereof, or seed comprises at least 2%more oleic acid and/or at least 2% less palmitic acid than the lipid inthe corresponding vegetative plant part, non-human organism or partthereof, or seed lacking the one or more exogenous polynucleotides,and/or

(viii) the total fatty acid content in the non-polar lipid of thevegetative plant part, non-human organism or part thereof, or seedcomprises at least 2% more oleic acid and/or at least 2% less palmiticacid than the non-polar lipid in the corresponding vegetative plantpart, non-human organism or part thereof, or seed lacking the one ormore exogenous polynucleotides,

(ix) the non-polar lipid(s) comprise a modified level of total sterols,preferably free sterols, steroyl esters and/or steroyl glycosides,

(x) the non-polar lipid(s) comprise waxes and/or wax esters, and

(xi) the non-human organism or part thereof is one member of apopulation or collection of at least about 1000 such non-human organismsor parts thereof.

In an embodiment, the one or more exogenous polynucleotide(s) comprisethe first exogenous polynucleotide and the second exogenouspolynucleotide, and wherein one or more or all of the features (ii) to(xi) apply.

In an embodiment of (ii) above, the total non-polar lipid content isbetween 5% and 25%, between 7% and 25%, between 10% and 25%, between 12%and 25%, between 15% and 25%, between 7% and 20%, between 10% and 20%,about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about16%, about 17%, about 18%, about 20%, or about 22%, each as a percentageof dry weight. In a more preferred embodiment, the vegetative plant partis a leaf portion having a surface area of at least 1 cm².

In preferred embodiments, the non-human organism or part thereof is aplant, an alga or an organism suitable for fermentation such as afungus. The part of the non-human organism may be a seed, fruit, or avegetative part of a plant such as an aerial plant part or a green partsuch as a leaf or stem. In another embodiment, the part is a cell of amulticellular organism. With respect to the part of the non-humanorganism, the part comprises at least one cell of the non-humanorganism. In further preferred embodiments, the non-human organism orpart thereof is further defined by features as defined in any of theembodiments described in the first and second aspects of the invention,including but not limited to Features (i), (ii) and (iii), and theexogenous polynucleotides or combinations of exogenous polynucleotidesas defined in any of the embodiments described in the first and secondaspects of the invention.

In an embodiment, the plant, vegetative plant part, non-human organismor part thereof, or seed comprises one or more exogenous polynucleotideswhich encode:

i) a Wrinkled 1 (WRI1) transcription factor and a DGAT,

ii) a WRI1 transcription factor and a DGAT and an Oleosin,

iii) a WRI1 transcription factor, a DGAT, a MGAT and an Oleosin,

iv) a monoacylglycerol acyltransferase (MGAT),

v) a diacylglycerol acyltransferase 2 (DGAT2),

vi) a MGAT and a glycerol-3-phosphate acyltransferase (GPAT),

vii) a MGAT and a DGAT,

viii) a MGAT, a GPAT and a DGAT,

ix) a WRI1 transcription factor and a MGAT,

x) a WRI1 transcription factor, a DGAT and a MGAT,

xi) a WRI1 transcription factor, a DGAT, a MGAT, an Oleosin and a GPAT,

xii) a DGAT and an Oleosin, or

xiii) a MGAT and an Oleosin, and

xiv) optionally, a silencing suppressor polypeptide,

wherein each exogenous polynucleotide is operably linked to a promoterwhich is capable of directing expression of the polynucleotide in aplant, vegetative plant part, non-human organism or part thereof, orseed, respectively. The one or more exogenous polynucleotides maycomprise nucleotides whose sequence is defined herein. Preferably, theplant, vegetative plant part, non-human organism or part thereof, orseed is homozygous for the one or more exogenous polynucleotides.Preferably, the exogenous polynucleotides are integrated into the genomeof the plant, vegetative plant part, non-human organism or part thereof,or seed. The one or more polynucleotides may be provided as separatemolecules or may be provided as a contiguous single molecule.Preferably, the exogenous polynucleotides are integrated in the genomeof the plant or organism at a single genetic locus or genetically linkedloci, more preferably in the homozygous state. More preferably, theintegrated exogenous polynucleotides are genetically linked with aselectable marker gene such as an herbicide tolerance gene.

In a preferred embodiment, the vegetative plant part, the non-humanorganism or part thereof, or the seed comprises a first exogenouspolynucleotide encoding a WRI1 and a second exogenous polynucleotideencoding a DGAT, preferably a DGAT1.

In another preferred embodiment, the vegetative plant part, thenon-human organism or part thereof, or the seed comprises a firstexogenous polynucleotide encoding a WRI1, a second exogenouspolynucleotide encoding a DGAT, preferably a DGAT1, and a thirdexogenous polynucleotide encoding an oleosin.

In a further embodiment, the vegetative plant part, the non-humanorganism or part thereof, or the seed comprises a first exogenouspolynucleotide encoding a WRI1, a second exogenous polynucleotideencoding a DGAT, preferably a DGAT1, a third exogenous polynucleotideencoding an oleosin, and a fourth exogenous polynucleotide encoding anMGAT, preferably an MGAT2.

In a further embodiment, the vegetative plant part, the non-humanorganism or part thereof, or the seed comprises a first exogenouspolynucleotide encoding a WRI1, a second exogenous polynucleotideencoding a DGAT, preferably a DGAT1, a third exogenous polynucleotideencoding an oleosin, and a fourth exogenous polynucleotide encoding LEC2or BBM.

In a further embodiment, the vegetative plant part, the non-humanorganism or part thereof, or the seed comprises a first exogenouspolynucleotide encoding a WRI1, a second exogenous polynucleotideencoding a DGAT, preferably a DGAT1, a third exogenous polynucleotideencoding an oleosin, a fourth exogenous polynucleotide encoding an MGAT,preferably an MGAT2, and a fifth exogenous polynucleotide encoding LEC2or BBM.

In a further embodiment, the vegetative plant part, the non-humanorganism or part thereof, or the seed comprises a first exogenouspolynucleotide encoding a WRI1, a second exogenous polynucleotideencoding a DGAT, preferably a DGAT1, a third exogenous polynucleotideencoding an oleosin, and a fourth exogenous polynucleotide encoding anRNA molecule which inhibits expression of a gene encoding a lipase suchas a CGi58 polypeptide.

In a further embodiment, the vegetative plant part, the non-humanorganism or part thereof, or the seed comprises a first exogenouspolynucleotide encoding a WRI1, a second exogenous polynucleotideencoding a DGAT, preferably a DGAT1, a third exogenous polynucleotideencoding an oleosin, a fourth exogenous polynucleotide encoding an RNAmolecule which inhibits expression of a gene encoding a lipase such as aCGi58 polypeptide, and a fifth exogenous polynucleotide encoding LEC2 orBBM.

In a further embodiment, the vegetative plant part, the non-humanorganism or part thereof, or the seed comprises a first exogenouspolynucleotide encoding a WRI1, a second exogenous polynucleotideencoding a DGAT, preferably a DGAT1, a third exogenous polynucleotideencoding an oleosin, a fourth exogenous polynucleotide encoding an RNAmolecule which inhibits expression of a gene encoding a lipase such as aCGi58 polypeptide, and a fifth exogenous polynucleotide encoding anMGAT, preferably an MGAT2.

In a further embodiment, the vegetative plant part, the non-humanorganism or part thereof, or the seed comprises a first exogenouspolynucleotide encoding a WRI1, a second exogenous polynucleotideencoding a DGAT, preferably a DGAT1, a third exogenous polynucleotideencoding an oleosin, a fourth exogenous polynucleotide encoding an RNAmolecule which inhibits expression of a gene encoding a lipase such as aCGi58 polypeptide, a fifth exogenous polynucleotide encoding an MGAT,preferably an MGAT2, and a sixth exogenous polynucleotide encoding LEC2or BBM.

In an embodiment, the seed comprises a first exogenous polynucleotideencoding a WRI1, a second exogenous polynucleotide encoding a DGAT,preferably a DGAT1, a third exogenous polynucleotide encoding anoleosin, and a fourth exogenous polynucleotide encoding an MGAT,preferably an MGAT2. Preferably, the seed further comprises a fifthexogenous polynucleotide encoding a GPAT.

Where relevant, instead of a polynucleotide encoding an RNA moleculewhich inhibits expression of a gene encoding a lipase such as a CGi58polypeptide, the vegetative plant part, the non-human organism or partthereof, or the seed has one or more introduced mutations in the lipasegene such as a CGi58 gene which confers reduced levels of the lipasepolypeptide when compared to an isogenic vegetative plant part,non-human organism or part thereof, or seed lacking the mutation.

In a preferred embodiment, the exogenous polynucleotides encoding theDGAT and oleosin are operably linked to a constitutive promoter, or apromoter active in green tissues of a plant at least before and up untilflowering, which is capable of directing expression of thepolynucleotides in the vegetative plant part, the non-human organism orpart thereof, or the seed. In a further preferred embodiment, theexogenous polynucleotide encoding WRI1, and RNA molecule which inhibitsexpression of a gene encoding a lipase such as a CGi58 polypeptide, isoperably linked to a constitutive promoter, a promoter active in greentissues of a plant at least before and up until flowering, or aninducible promoter, which is capable of directing expression of thepolynucleotides in the vegetative plant part, the non-human organism orpart thereof, or the seed. In yet a further preferred embodiment, theexogenous polynucleotides encoding LEC2, BBM and/or MGAT2 are operablylinked to an inducible promoter which is capable of directing expressionof the polynucleotides in the vegetative plant part, the non-humanorganism or part thereof, or the seed.

In each of the above embodiments, the total non-polar lipid content ofthe vegetative plant part, or non-human organism or part thereof, or theseed, preferably a plant leaf or part thereof, stem or tuber, is atleast about 3%, more preferably at least about 5%, preferably at leastabout 7%, more preferably at least about 10%, more preferably at leastabout 11%, more preferably at least about 12%, more preferably at leastabout 13%, more preferably at least about 14%, or more preferably atleast about 15% (w/w dry weight or seed weight). In a further preferredembodiment, the total non-polar lipid content is between 5% and 25%,between 7% and 25%, between 10% and 25%, between 12% and 25%, between15% and 25%, between 7% and 20%, between 10% and 20%, between 10% and15%, between 15% and 20%, between 20% and 25%, about 10%, about 11%,about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about18%, about 20%, or about 22%, each as a percentage of dry weight or seedweight. In a particularly preferred embodiment, the vegetative plantpart is a leaf (or leaves) or a portion thereof. In a more preferredembodiment, the vegetative plant part is a leaf portion having a surfacearea of at least 1 cm².

Furthermore, in each of the above embodiments, the total TAG content ofthe vegetative plant part, or non-human organism or part thereof, or theseed, preferably a plant leaf or part thereof, stem or tuber, is atleast about 3%, more preferably at least about 5%, preferably at leastabout 7%, more preferably at least about 10%, more preferably at leastabout 11%, more preferably at least about 12%, more preferably at leastabout 13%, more preferably at least about 14%, more preferably at leastabout 15%, or more preferably at least about 17% (w/w dry weight or seedweight). In a further preferred embodiment, the total TAG content isbetween 5% and 30%, between 7% and 30%, between 10% and 30%, between 12%and 30%, between 15% and 30%, between 7% and 30%, between 10% and 30%,between 20% and 28%, between 18% and 25%, between 22% and 30%, about10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%,about 17%, about 18%, about 20%, or about 22%, each as a percentage ofdry weight or seed weight. In a particularly preferred embodiment, thevegetative plant part is a leaf (or leaves) or a portion thereof. In amore preferred embodiment, the vegetative plant part is a leaf portionhaving a surface area of at least 1 cm².

Furthermore, in each of the above embodiments, the total lipid contentof the vegetative plant part, or non-human organism or part thereof, orthe seed, preferably a plant leaf or part thereof, stem or tuber, is atleast about 3%, more preferably at least about 5%, preferably at leastabout 7%, more preferably at least about 10%, more preferably at leastabout 11%, more preferably at least about 12%, more preferably at leastabout 13%, more preferably at least about 14%, more preferably at leastabout 15%, more preferably at least about 17% (w/w dry weight or seedweight), more preferably at least about 20%, more preferably at leastabout 25%. In a further preferred embodiment, the total lipid content isbetween 5% and 35%, between 7% and 35%, between 10% and 35%, between 12%and 35%, between 15% and 35%, between 7% and 35%, between 10% and 20%,between 18% and 28%, between 20% and 28%, between 22% and 28%, about10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%,about 17%, about 18%, about 20%, about 22%, or about 25%, each as apercentage of dry weight or seed weight. Typically, the total lipidcontent of the vegetative plant part, or non-human organism or partthereof is about 2-3% higher than the non-polar lipid content. In aparticularly preferred embodiment, the vegetative plant part is a leaf(or leaves) or a portion thereof. In a more preferred embodiment, thevegetative plant part is a leaf portion having a surface area of atleast 1 cm².

In an embodiment, the vegetative plant part, the non-human organism orpart thereof, or the seed, preferably the vegetative plant part,comprises a first exogenous polynucleotide encoding a WRI1, a secondexogenous polynucleotide encoding a DGAT, preferably a DGAT1, a thirdexogenous polynucleotide encoding an MGAT, preferably an MGAT2, and afourth exogenous polynucleotide encoding an oleosin, wherein thevegetative plant part, non-human organism or part thereof, or seed hasone or more or all of the following features:

i) a total lipid content of at least 8%, at least 10%, at least 12%, atleast 14%, or at least 15.5% (% weight of dry weight or seed weight),

ii) at least a 3 fold, at least a 5 fold, at least a 7 fold, at least an8 fold, or least a 10 fold, at higher total lipid content in thevegetative plant part or non-human organism relative to a correspondingvegetative plant part or non-human organism lacking the exogenouspolynucleotides,

iii) a total TAG content of at least 5%, at least 6%, at least 6.5% orat least 7% (% weight of dry weight or seed weight),

iv) at least a 40 fold, at least a 50 fold, at least a 60 fold, or atleast a 70 fold, or at least a 100 fold, higher total TAG contentrelative to a corresponding vegetative plant part or non-human organismlacking the exogenous polynucleotides,

v) oleic acid comprises at least 15%, at least 19% or at least 22% (%weight) of the fatty acids in TAG,

vi) at least a 10 fold, at least a 15 fold or at least a 17 fold higherlevel of oleic acid in TAG relative to a corresponding vegetative plantpart or non-human organism lacking the exogenous polynucleotides,

vii) palmitic acid comprises at least 20%, at least 25%, at least 30% orat least 33% (% weight) of the fatty acids in TAG,

viii) at least a 1.5 fold higher level of palmitic acid in TAG relativeto a corresponding vegetative plant part on non-human organism lackingthe exogenous polynucleotides,

ix) linoleic acid comprises at least 22%, at least 25%, at least 30% orat least 34% (% weight) of the fatty acids in TAG,

x) α-linolenic acid comprises less than 20%, less than 15%, less than11% or less than 8% (% weight) of the fatty acids in TAG, and

xi) at least a 5 fold, or at least an 8 fold, lower level of α-linolenicacid in TAG relative to a corresponding vegetative plant part ornon-human organism lacking the exogenous polynucleotides. In thisembodiment, preferably the vegetative plant part at least hasfeature(s), i), ii) iii), iv), i) and ii), i) and iii), i) and iv), i)to iii), i), iii) and iv), i) to iv), ii) and iii), ii) and iv), ii) toiv), or iii) and iv). In an embodiment, % dry weight is % leaf dryweight.

In a further embodiment, the vegetative plant part, the non-humanorganism or part thereof, or the seed, preferably the vegetative plantpart, comprises a first exogenous polynucleotide encoding a WRI1, asecond exogenous polynucleotide encoding a DGAT, preferably a DGAT1, athird exogenous polynucleotide encoding an oleosin, wherein thevegetative plant part, non-human organism or part thereof, or seed hasone or more or all of the following features:

i) a total TAG content of at least 10%, at least 12.5%, at least 15% orat least 17% (% weight of dry weight or seed weight),

ii) least a 40 fold, at least a 50 fold, at least a 60 fold, or at leasta 70 fold, or at least a 100 fold, higher total TAG content in thevegetative plant part or non-human organism relative to a correspondingvegetative plant part or non-human organism lacking the exogenouspolynucleotides,

iii) oleic acid comprises at least 19%, at least 22%, or at least 25% (%weight) of the fatty acids in TAG,

iv) at least a 10 fold, at least a 15 fold, at least a 17 fold, or atleast a 19 fold, higher level of oleic acid in TAG in the vegetativeplant part or non-human organism relative to a corresponding vegetativeplant part or non-human organism lacking the exogenous polynucleotides,

v) palmitic acid comprises at least 20%, at least 25%, or at least 28%(% weight) of the fatty acids in TAG,

vi) at least a 1.25 fold higher level of palmitic acid in TAG in thevegetative plant part or non-human organism relative to a correspondingvegetative plant part or non-human organism lacking the exogenouspolynucleotides,

vii) linoleic acid comprises at least 15%, or at least 20%, (% weight)of the fatty acids in TAG,

viii) α-linolenic acid comprises less than 15%, less than 11% or lessthan 8% (% weight) of the fatty acids in TAG, and

ix) at least a 5 fold, or at least an 8 fold, lower level of α-linolenicacid in TAG in the vegetative plant part or non-human organism relativeto a corresponding vegetative plant part or non-human organism lackingthe exogenous polynucleotides. In this embodiment, preferably thevegetative plant part at least has feature(s), i), ii), or i) and ii).In an embodiment, % dry weight is % leaf dry weight.

Preferably, the defined features for the two above embodiments are as atthe flowering stage of the plant.

In a fourth aspect, the invention provides a plant seed capable ofgrowing into a plant of the invention, or obtained from a plant of theinvention, for example a non-human organism of the invention which is aplant. In an embodiment, the seed comprises one or more exogenouspolynucleotides as defined herein.

In a fifth aspect, the invention provides a process for obtaining a cellwith enhanced ability to produce one or more non-polar lipids, theprocess comprising the steps of:

-   -   a) introducing into a cell one or more exogenous        polynucleotides,    -   b) expressing the one or more exogenous polynucleotides in the        cell or a progeny cell thereof,    -   c) analysing the lipid content of the cell or progeny cell, and    -   d) selecting a cell or progeny cell having an increased level of        one or more non-polar lipids relative to a corresponding cell or        progeny cell lacking the exogenous polynucleotides,        wherein the one or more exogenous polynucleotides encode

i) a Wrinkled 1 (WRI1) transcription factor and a DGAT,

ii) a WRI1 transcription factor and a DGAT and an Oleosin,

iii) a WRI1 transcription factor, a DGAT, a MGAT and an Oleosin,

iv) a monoacylglycerol acyltransferase (MGAT),

v) a diacylglycerol acyltransferase 2 (DGAT2),

vi) a MGAT and a glycerol-3-phosphate acyltransferase (GPAT),

vii) a MGAT and a DGAT,

viii) a MGAT, a GPAT and a DGAT,

ix) a WRI1 transcription factor and a MGAT,

x) a WRI1 transcription factor, a DGAT and a MGAT,

xi) a WRI1 transcription factor, a DGAT, a MGAT, an Oleosin and a GPAT,

xii) a DGAT and an Oleosin, or

xiii) a MGAT and an Oleosin, and

xiv) optionally, a silencing suppressor polypeptide,

wherein each exogenous polynucleotide is operably linked to a promoterthat is capable of directing expression of the exogenous polynucleotidein the cell or progeny cell.

In an embodiment, the selected cell or progeny cell comprises:

i) a first exogenous polynucleotide encoding a WRI1 and a secondexogenous polynucleotide encoding a DGAT, preferably a DGAT1,

ii) a first exogenous polynucleotide encoding a WRI1, a second exogenouspolynucleotide encoding a DGAT, preferably a DGAT1, and a thirdexogenous polynucleotide encoding an oleosin,

iii) a first exogenous polynucleotide encoding a WRI1, a secondexogenous polynucleotide encoding a DGAT, preferably a DGAT1, a thirdexogenous polynucleotide encoding an oleosin, and a fourth exogenouspolynucleotide encoding an MGAT, preferably an MGAT2,

iv) a first exogenous polynucleotide encoding a WRI1, a second exogenouspolynucleotide encoding a DGAT, preferably a DGAT1, a third exogenouspolynucleotide encoding an oleosin, and a fourth exogenouspolynucleotide encoding LEC2 or BBM,

v) a first exogenous polynucleotide encoding a WRI1, a second exogenouspolynucleotide encoding a DGAT, preferably a DGAT1, a third exogenouspolynucleotide encoding an oleosin, a fourth exogenous polynucleotideencoding an MGAT, preferably an MGAT2, and a fifth exogenouspolynucleotide encoding LEC2 or BBM,

vi) a first exogenous polynucleotide encoding a WRI1, a second exogenouspolynucleotide encoding a DGAT, preferably a DGAT1, a third exogenouspolynucleotide encoding an oleosin, and a fourth exogenouspolynucleotide encoding an RNA molecule which inhibits expression of agene encoding a lipase such as a CGi58 polypeptide,

vii) a first exogenous polynucleotide encoding a WRI1, a secondexogenous polynucleotide encoding a DGAT, preferably a DGAT1, a thirdexogenous polynucleotide encoding an oleosin, a fourth exogenouspolynucleotide encoding an RNA molecule which inhibits expression of agene encoding a lipase such as a CGi58 polypeptide, and a fifthexogenous polynucleotide encoding LEC2 or BBM,

viii) a first exogenous polynucleotide encoding a WRI1, a secondexogenous polynucleotide encoding a DGAT, preferably a DGAT1, a thirdexogenous polynucleotide encoding an oleosin, a fourth exogenouspolynucleotide encoding an RNA molecule which inhibits expression of agene encoding a lipase such as a CGi58 polypeptide, and a fifthexogenous polynucleotide encoding an MGAT, preferably an MGAT2, or

ix) a first exogenous polynucleotide encoding a WRI1, a second exogenouspolynucleotide encoding a DGAT, preferably a DGAT1, a third exogenouspolynucleotide encoding an oleosin, a fourth exogenous polynucleotideencoding an RNA molecule which inhibits expression of a gene encoding alipase such as a CGi58 polypeptide, a fifth exogenous polynucleotideencoding an MGAT, preferably an MGAT2, and a sixth exogenouspolynucleotide encoding LEC2 or BBM.

In a further embodiment, the selected cell or progeny cell is a cell ofa plant seed and comprises a first exogenous polynucleotide encoding aWRI1, a second exogenous polynucleotide encoding a DGAT, preferably aDGAT1, a third exogenous polynucleotide encoding an oleosin, and afourth exogenous polynucleotide encoding an MGAT, preferably an MGAT2.Preferably, the seed further comprises a fifth exogenous polynucleotideencoding a GPAT.

In a preferred embodiment, the one or more exogenous polynucleotides arestably integrated into the genome of the cell or progeny cell.

In a preferred embodiment, the process further comprises a step ofregenerating a transgenic plant from the cell or progeny cell comprisingthe one or more exogenous polynucleotides. The step of regenerating atransgenic plant may be performed prior to the step of expressing theone or more exogenous polynucleotides in the cell or a progeny cellthereof, and/or prior to the step of analysing the lipid content of thecell or progeny cell, and/or prior to the step of selecting the cell orprogeny cell having an increased level of one or more non-polar lipids.The process may further comprise a step of obtaining seed or a progenyplant from the transgenic plant, wherein the seed or progeny plantcomprises the one or more exogenous polynucleotides.

The process of the fifth aspect may be used as a screening assay todetermine whether a polypeptide encoded by an exogenous polynucleotidehas a desired function. The one or more exogenous polynucleotides inthis aspect may comprise a sequence as defined above. Further, the oneor more exogenous polynucleotides may not be known prior to the processto encode a WRI1 transcription factor and a DGAT, a WRI1 transcriptionfactor and a MGAT, a WRI1 transcription factor, a DGAT and a MGAT, aWRI1 transcription factor, a DGAT, a MGAT and an Oleosin, a WRI1transcription factor, a DGAT, a MGAT, an Oleosin and a GPAT, a WRI1transcription factor, a DGAT and an oleosin, a DGAT and an Oleosin, or aMGAT and an Oleosin, but rather may be candidates therefor. The processtherefore may be used as an assay to identify or select polynucleotidesencoding a WRI1 transcription factor and a DGAT, a WRI1 transcriptionfactor and a MGAT, a WRI1 transcription factor, a DGAT and a MGAT, aWRI1 transcription factor, a DGAT, a MGAT and an Oleosin, a WRI1transcription factor, a DGAT, a MGAT, an Oleosin and a GPAT, a WRI1transcription factor, a DGAT and an oleosin, a DGAT and an Oleosin, or aMGAT and an Oleosin. The candidate polynucleotides are introduced into acell and the products analysed to determine whether the candidates havethe desired function.

In a sixth aspect, the invention provides a transgenic cell ortransgenic plant obtained using a process of the invention, or avegetative plant part or seed obtained therefrom which comprises the oneor more exogenous polynucleotides.

In a seventh aspect, the invention provides a use of one or morepolynucleotides encoding, or a genetic construct comprisingpolynucleotides encoding:

i) a Wrinkled 1 (WRI1) transcription factor and a DGAT,

ii) a WRI1 transcription factor and a DGAT and an Oleosin,

iii) a WRI1 transcription factor, a DGAT, a MGAT and an Oleosin,

iv) a monoacylglycerol acyltransferase (MGAT),

v) a diacylglycerol acyltransferase 2 (DGAT2),

vi) a MGAT and a glycerol-3-phosphate acyltransferase (GPAT),

vii) a MGAT and a DGAT,

viii) a MGAT, a GPAT and a DGAT,

ix) a WRI1 transcription factor and a MGAT,

x) a WRI1 transcription factor, a DGAT and a MGAT,

xi) a WRI1 transcription factor, a DGAT, a MGAT, an Oleosin and a GPAT,

xii) a DGAT and an Oleosin, or

xiii) a MGAT and an Oleosin, and

xiv) optionally, a silencing suppressor polypeptide,

for producing a transgenic cell, a transgenic non-human organism or apart thereof or a transgenic seed having an enhanced ability to produceone or more non-polar lipids relative to a corresponding cell, non-humanorganism or part thereof, or seed lacking the one or morepolynucleotides, wherein each of the one or more polynucleotides isexogenous to the cell, non-human organism or part thereof, or seed andis operably linked to a promoter which is capable of directingexpression of the polynucleotide in a cell, a non-human organism or apart thereof or a seed, respectively.

In an embodiment, the invention provides a use of a first polynucleotideencoding an RNA or transcription factor polypeptide that increases theexpression of one or more glycolytic or fatty acid biosynthetic genes ina cell, a non-human organism or a part thereof, or a seed, together witha second polynucleotide that encodes an RNA or polypeptide involved inbiosynthesis of one or more non-polar lipids, for producing a transgeniccell, a transgenic non-human organism or part thereof, or a transgenicseed having an enhanced ability to produce one or more non-polar lipidsrelative to a corresponding cell, non-human organism or part thereof, orseed lacking the first and second polynucleotides, wherein the first andsecond polynucleotides are each exogenous to the cell, non-humanorganism or part thereof, or seed and are each operably linked to apromoter which is capable of directing expression of the polynucleotidein the transgenic cell, transgenic non-human organism or part thereof,or transgenic seed, respectively.

In a further embodiment, the invention provides a use of one or morepolynucleotides for producing a transgenic cell, a transgenic non-humanorganism or part thereof, or a transgenic seed having an enhancedability to produce one or more non-polar lipid(s) relative to acorresponding cell, non-human organism or part thereof, or seed lackingthe one or more exogenous polynucleotides, wherein each of the one ormore polynucleotides is exogenous to the cell, non-human organism orpart thereof, or seed and is operably linked to a promoter which iscapable of directing expression of the polynucleotide in a cell, anon-human organism or a part thereof, or a seed, respectively, andwherein the non-polar lipid(s) comprise a fatty acid which comprises ahydroxyl group, an epoxy group, a cyclopropane group, a doublecarbon-carbon bond, a triple carbon-carbon bond, conjugated doublebonds, a branched chain such as a methylated or hydroxylated branchedchain, or a combination of two or more thereof, or any of two, three,four, five or six of the aforementioned groups, bonds or branchedchains. Such uses also have utility as screening assays.

In an eighth aspect, the invention provides a process for producingseed, the process comprising:

i) growing a plant, multiple plants, or non-human organism according tothe invention, and

ii) harvesting seed from the plant, plants, or non-human organism.

In a preferred embodiment, the process comprises growing a population ofat least about 1000 such plants in a field, and harvesting seed from thepopulation of plants. The harvested seed may be placed in a containerand transported away from the field, for example exported out of thecountry, or stored prior to use.

In a ninth aspect, the invention provides a fermentation processcomprising the steps of:

i) providing a vessel containing a liquid composition comprising anon-human organism of the invention which is suitable for fermentation,and constituents required for fermentation and fatty acid biosynthesis,and

ii) providing conditions conducive to the fermentation of the liquidcomposition contained in said vessel.

In a tenth aspect, the invention provides a recovered or extracted lipidobtainable by a process of the invention, or obtainable from avegetative plant part, non-human organism or part thereof, cell orprogeny cell, transgenic plant, or seed of the invention. The recoveredor extracted lipid, preferably oil such as seedoil, may have an enhancedTAG content, DAG content, TAG and DAG content, MAG content, PUFAcontent, specific PUFA content, or a specific fatty acid content, and/ortotal non-polar lipid content. In a preferred embodiment, the MAG is2-MAG. The extent of the increased TAG content, DAG content, TAG and DAGcontent, MAG content, PUFA content, specific PUFA content, specificfatty acid content and/or total non-polar lipid content may be asdefined in Feature (i).

In an eleventh aspect, the invention provides an industrial productproduced by a process of the invention, preferably which is ahydrocarbon product such as fatty acid esters, preferably fatty acidmethyl esters and/or a fatty acid ethyl esters, an alkane such asmethane, ethane or a longer-chain alkane, a mixture of longer chainalkanes, an alkene, a biofuel, carbon monoxide and/or hydrogen gas, abioalcohol such as ethanol, propanol, or butanol, biochar, or acombination of carbon monoxide, hydrogen and biochar.

In a twelfth aspect, the invention provides a use of a plant, vegetativeplant part, non-human organism or a part thereof, cell or progeny cell,transgenic plant produced by a process of the invention, or a seed or arecovered or extracted lipid of the invention for the manufacture of anindustrial product. The industrial product may be as defined above.

In a thirteenth aspect, the invention provides a process for producingfuel, the process comprising:

i) reacting a lipid of the invention with an alcohol, optionally in thepresence of a catalyst, to produce alkyl esters, and

ii) optionally, blending the alkyl esters with petroleum based fuel. Thealkyl esters are preferably methyl esters. The fuel produced by theprocess may comprise a minimum level of the lipid of the invention or ahydrocarbon product produced therefrom such as at least 10%, at least20%, or at least 30% by volume.

In a fourteenth aspect, the invention provides a process for producing asynthetic diesel fuel, the process comprising:

i) converting lipid in a vegetative plant, non-human organism or partthereof of the invention to a syngas by gasification, and

ii) converting the syngas to a biofuel using a metal catalyst or amicrobial catalyst.

In a fifteenth aspect, the invention provides a process for producing abiofuel, the process comprising converting lipid in a vegetative plantpart, non-human organism or part thereof of the invention to bio-oil bypyrolysis, a bioalcohol by fermentation, or a biogas by gasification oranaerobic digestion.

In a sixteenth aspect, the invention provides a process for producing afeedstuff, the process comprising admixing a plant, vegetative plantpart thereof, non-human organism or part thereof, cell or progeny cell,transgenic plant produced by a process of the invention, seed, recoveredor extracted lipid, or an extract or portion thereof, with at least oneother food ingredient.

In a seventeenth aspect, the invention provides feedstuffs, cosmetics orchemicals comprising a plant, vegetative part thereof, non-humanorganism or part thereof, cell or progeny cell, transgenic plantproduced by a process of the invention, seed, or a recovered orextracted lipid of the invention, or an extract or portion thereof.

Naturally, when vegetative material of a plant is to be harvestedbecause of its oil content it is desirable to harvest the material whenlipid levels are as high as possible. The present inventors have notedan association between the glossiness of the vegetative tissue of theplants of the invention and oil content, with high levels of lipid beingassociated with high gloss. Thus, the glossiness of the vegetativematerial can be used as marker to assist in determining when to harvestthe material.

In a further aspect, the invention provides a recombinant cellcomprising one or more exogenous polynucleotide(s) and an increasedlevel of one or more non-polar lipid(s) relative to a corresponding celllacking the one or more exogenous polynucleotide(s),

wherein each of the one or more exogenous polynucleotides is operablylinked to a promoter which is capable of directing expression of thepolynucleotide in a cell, and wherein one or more or all of thefollowing features apply:

(a) the one or more exogenous polynucleotide(s) comprise a firstexogenous polynucleotide which encodes an RNA or transcription factorpolypeptide that increases the expression of one or more glycolytic orfatty acid biosynthetic genes in a non-human organism or a part thereof,and a second exogenous polynucleotide which encodes an RNA orpolypeptide involved in biosynthesis of one or more non-polar lipids,

(b) if the cell is a cell of a vegetative part of a plant, the cell hasa total non-polar lipid content of at least about 3%, more preferably atleast about 5%, preferably at least about 7%, more preferably at leastabout 10%, more preferably at least about 11%, more preferably at leastabout 12%, more preferably at least about 13%, more preferably at leastabout 14%, or more preferably at least about 15% (w/w),

(c) the cell is an alga selected from the group consisting of diatoms(bacillariophytes), green algae (chlorophytes), blue-green algae(cyanophytes), golden-brown algae (chrysophytes), haptophytes, brownalgae and heterokont algae,

(d) the one or more non-polar lipid(s) comprise a fatty acid whichcomprises a hydroxyl group, an epoxy group, a cyclopropane group, adouble carbon-carbon bond, a triple carbon-carbon bond, conjugateddouble bonds, a branched chain such as a methylated or hydroxylatedbranched chain, or a combination of two or more thereof, or any of two,three, four, five or six of the aforementioned groups, bonds or branchedchains,

(e) the total fatty acid content in the non-polar lipid(s) comprises atleast 2% more oleic acid and/or at least 2% less palmitic acid than thenon-polar lipid(s) in the corresponding cell lacking the one or moreexogenous polynucleotides,

(f) the non-polar lipid(s) comprise a modified level of total sterols,preferably free (non-esterified) sterols, steroyl esters, steroylglycosides, relative to the non-polar lipid(s) in the corresponding celllacking the one or more exogenous polynucleotides,

(g) the non-polar lipid(s) comprise waxes and/or wax esters, and

(h) the cell is one member of a population or collection of at leastabout 1000 such cells.

In an embodiment, the one or more exogenous polynucleotide(s) comprisethe first exogenous polynucleotide and the second exogenouspolynucleotide, and wherein one or more or all of the features (b) to(h) apply.

In a further aspect, the present invention provides a method ofdetermining when to harvest a plant to optimize the amount of lipid inthe vegetative tissue of the plant at harvest, the method comprising

i) measuring the gloss of the vegetative tissue,

ii) comparing the measurement with a pre-determined minimum glossinesslevel, and

iii) optionally harvesting the plant.

In another aspect, the present invention provides a method of predictingthe quantity of lipid in vegetative tissue of a plant, the methodcomprising measuring the gloss of the vegetative tissue.

In a preferred embodiment of the two above aspects the vegetative tissueis a leaf (leaves) or a portion thereof.

In a further aspect, the present invention provides a method of tradinga plant or a part thereof, comprising obtaining the plant or partcomprising a cell of the invention, and trading the obtained plant orplant part for pecuniary gain.

In an embodiment, the method further comprises one or more or all of:

i) cultivating the plant,

ii) harvesting the plant part from the plant,

iii) storing the plant or part thereof, or

iv) transporting the plant or part thereof to a different location.

In a further aspect, the present invention provides a process forproducing bins of plant parts comprising:

a) harvesting plant parts comprising a cell of the invention bycollecting the plant parts from the plants, or by separating the plantparts from other parts of the plants,

b) optionally, sifting and/or sorting the harvested plant parts, and

c) loading the plant parts of a) or the sifted and/or sorted plant partsof b) into bins, thereby producing bins of the plant parts.

Any embodiment herein shall be taken to apply mutatis mutandis to anyother embodiment unless specifically stated otherwise.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended for the purpose ofexemplification only. Functionally-equivalent products, compositions andmethods are clearly within the scope of the invention, as describedherein.

Throughout this specification, unless specifically stated otherwise orthe context requires otherwise, reference to a single step, compositionof matter, group of steps or group of compositions of matter shall betaken to encompass one and a plurality (i.e. one or more) of thosesteps, compositions of matter, groups of steps or group of compositionsof matter.

The invention is hereinafter described by way of the followingnon-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1. A representation of various lipid synthesis pathways, most ofwhich converge at DAG, a central molecule in lipid synthesis. This modelincludes one possible route to the formation of sn-2 MAG which could beused by a bi-functional MGAT/DGAT for DAG formation fromglycerol-3-phosphate (G-3-P). Abbreviations are as follows:

-   -   G-3-P; glycerol-3-phosphate    -   LysoPA; lysophosphatidic acid    -   PA; phosphatidic acid    -   MAG; monoacylglycerol    -   DAG; diacylglycerol    -   TAG; triacylglycerol    -   Acyl-CoA and FA-CoA; acyl-coenzyme A and fatty acyl-coenzyme A    -   PC; phosphatidylcholine    -   GPAT; glycerol-3-phosphate acyltransferase; glycerol-3-phosphate        O-acyltransferase; acyl-CoA:sn-glycerol-3-phosphate        1-O-acyltransferase; EC 2.3.1.15    -   GPAT4; glycerol-3-phosphate acyltransferase 4    -   GPAT6; glycerol-3-phosphate acyltransferase 6    -   LPAAT; 1-acyl-glycerol-3-phosphate acyltransferase;        1-acylglycerol-3-phosphate O-acyltransferase;        acyl-CoA:1-acyl-sn-glycerol-3-phosphate 2-O-acyltransferase; EC        2.3.1.51    -   PAP; phosphatidic acid phosphatase; phosphatidate phosphatase;        phosphatic acid phosphohydrolase; phosphatidic acid phosphatase;        EC 3.1.3.4    -   MGAT; an acyltransferase having monoacylglycerol acyltransferase        (MGAT; 2-acylglycerol O-acyltransferase acyl-CoA:2-acylglycerol        O-acyltransferase; EC 2.3.1.22) activity    -   M/DGAT; an acyltransferase having monoacylglycerol        acyltransferase (MGAT; 2-acylglycerol O-acyltransferase;        acyl-CoA:2-acylglycerol O-acyltransferase; EC 2.3.1.22) and/or        diacylglycerol acyltransferase (DGAT; diacylglycerol        O-acyltransferase; acyl-CoA:1,2-diacyl-sn-glycerol        O-acyltransferase; EC 2.3.1.20) activity    -   LPCAT; acyl-CoA:lysophosphatidylcholine acyltransferase;        1-acylglycerophosphocholine O-acyltransferase;        acyl-CoA:1-acyl-sn-glycero-3-phosphocholine O-acyltransferase;        EC 2.3.1.23    -   PLD-Z; Phospholipase D zeta; choline phosphatase; lecithinase D;        lipophosphodiesterase II; EC 3.1.4.4    -   CPT; CDP-choline:diacylglycerol cholinephosphotransferase;        1-alkyl-2-acetylglycerol cholinephosphotransferase;        alkylacylglycerol cholinephosphotransferase;        cholinephosphotransferase; phosphorylcholine-glyceride        transferase; EC 2.7.8.2    -   PDCT; phosphatidylcholine:diacylglycerol        cholinephosphotransferase    -   PLC; phospholipase C; EC 3.1.4.3    -   PDAT; phospholipid:diacylglycerol acyltransferase;        phospholipid:1,2-diacyl-sn-glycerol O-acyltransferase; EC        2.3.1.158    -   Pi; inorganic phosphate

FIG. 2. Relative DAG and TAG increases in Nicotiana benthamiana leaftissue transformed with constructs encoding p19 (negative control),Arabidopsis thaliana DGAT1, Mus musculus MGAT1 and a combination ofDGAT1 and MGAT1, each expressed from the 35S promoter. The MGAT1 enzymewas far more active than the DGAT1 enzyme in promoting both DAG and TAGaccumulation in leaf tissue. Expression of the MGAT1 gene resulted intwice as much DAG and TAG accumulation in leaf tissue compared toexpression of DGAT1 alone.

FIG. 3. Relative TAG increases in N. benthamiana leaf transformed withconstructs encoding p19 (negative control), A. thaliana DGAT1, M.musculus MGAT2 and a combination of MGAT2 and DGAT1. Error bars denotestandard error of triplicate samples.

FIG. 4. Radioactivity (DPM) in MAG, DAG and TAG fractions isolated fromtransiently-transformed N. benthamiana leaf lysates fed withsn-2-MAG[¹⁴C] and unlabelled oleic acid over a time-course. Theconstructs used were as for FIG. 3.

FIG. 5. As for FIG. 4 but fed [¹⁴C]G-3-P and unlabelled oleic acid.

FIG. 6. Autoradiogram of TLC plate showing TAG-formation by A. thalianaDGAT1 and M. musculus MGAT1 but not M. musculus MGAT2 in yeast assays.The assay is described in Example 5.

FIG. 7. TAG levels in Arabidopsis thaliana T2 and T3 seeds transformedwith a chimeric DNA expressing MGAT2 relative to parental(untransformed) control. Seeds were harvested at maturity (dessicated).SW: desiccated seed weight. TAG levels are given as μg TAG per 100 μgseed weight.

FIG. 8. Total fatty acid content in seed of transformed Arabidopsisthaliana plants transformed with constructs encoding MGAT1 or MGAT2.

FIG. 9. Relative TAG level in transiently-transformed N. benthamianaleaf tissue compared to Arabidopsis thaliana DGAT1 overexpression.

FIG. 10. TAG conversion from sn-1,2-DAG in DGAT assay from microsomes ofN. benthamiana leaf tissues expressing P19 control, Arabidopsis thalianaDGAT1 and Arabidopsis thaliana DGAT2

FIG. 11. Total FAME quantification in A. thaliana seeds transformed withpJP3382 and pJP3383.

FIG. 12. Maximum TAG levels obtained for different gene combinationstransiently expressed in N. benthamiana leaves. The V2 negative controlrepresents the average TAG level based on 15 independent repeats.

FIG. 13. Co-expression of the genes coding for the Arabidopsis thalianaDGAT1 acyltransferase and A. thaliana WRI1 transcription factor resultedin a synergistic effect on TAG levels in Nicotiana benthamiana leaves.Data shown are averages and standard deviations of five independentinfiltrations.

FIG. 14 TAG levels in stably-transformed N. benthamiana aerial seedlingtissue. Total lipids were extracted from aerial tissues of N.benthamiana seedlings and analysed by TLC-FID using an internal DAGEstandard to allow accurate comparison between samples.

FIG. 15. Total fatty acid levels of A. thaliana T2 seed populationstransformed with control vector (pORE04), M. musculus MGAT1 (35S:MGAT1)or M. musculus MGAT2 (35S:MGAT2).

FIG. 16. Map of the insertion region between the left and right bordersof pJP3502. TER Glyma-Lectin denotes the Glycine max lectin terminator;Arath-WRI1, Arabidopsis thaliana WRI1 transcription factor codingregion; PRO Arath-Rubisco SSU, A. thaliana rubisco small subunitpromoter; Sesin-Oleosin, Sesame indicum oleosin coding region; PROCaMV35S-Ex2, cauliflower mosaic virus 35S promoter having a duplicatedenhancer region; Arath-DGAT1, A. thaliana DGAT1 acyltransferase codingregion; TER Agrtu-NOS, Agrobacterium tumefaciens nopaline synthaseterminator.

FIG. 17. Schematic representation of the construct pJP3503 including theinsertion region between the left and right borders of pJP3503. TERAgrtu-NOS denotes the Agrobacterium tumefaciens nopaline synthaseterminator; Musmu-MGAT2, Mus Musculus MGAT2 acyltransferase; PROCaMV24S-Ex2, cauliflower mosaic virus 35S duplicated enhancer region;TER Glyma-Lectin, Glycine max lectin terminator; Arath-WRI1, Arabidopsisthaliana WRI1 transcription factor; PRO Arath-Rubisco SSU, A. thalianarubisco small subunit promoter; Sesin-Oleosin, Sesame indicum oleosin;Arath-DGAT1, A. thaliana DGAT1 acyltransferase

FIG. 18. TAG yields in different aged leaves of three wild type tobaccoplants (wt1-3) and three pJP3503 primary transformants (4, 29, 21). Leafstages are indicated by ‘G’, green; ‘YG’, yellow-green; ‘Y’, yellow.Plant stages during sampling were budding, wild type 1; first flowersappearing, wild type 2; flowering, wild type 3; producing seed pods(pJP3503 transformants).

FIG. 19A. DNA insert containing expression cassettes for the Umbelopsisramanniana DGAT2A expressed by the Glycine max alpha′ subunitbeta-conglycinin promoter, Arabidopsis thaliana WRI1 expressed by theGlycine max kunitz trypsin inhibitor 3 promoter and the Mus musculusMGAT2 expressed by the Glycine max alpha′ subunit beta-conglycininpromoter. Gene coding regions and expression cassettes are excisable byrestriction digestion.

FIG. 19B. DNA insert containing expression cassettes for the Arabidopsisthaliana LEC2 and WRI1 transcription factor genes expressed by inducibleAspergillus alcA promoters, the Arabidopsis thaliana DGAT1 expressed bythe constitutive CaMV-35S promoter and the Aspergillus alcR geneexpressed by the constitutive CsVMV promoter. Expressed of the LEC2 andWRI1 transcription factors is induced by ethanol or an analagouscompound.

FIG. 20. pJP3507 map

FIG. 21. pJP3569 map

KEY TO THE SEQUENCE LISTING

SEQ ID NO:1 Mus musculus codon optimised MGAT1

SEQ ID NO:2 Mus musculus codon optimised MGAT2

SEQ ID NO:3 Ciona intestinalis codon optimised MGAT1

SEQ ID NO:4 Tribolium castaneum codon optimised MGAT1

SEQ ID NO:5 Danio rerio codon optimised MGAT1

SEQ ID NO:6 Danio rerio codon optimised MGAT2

SEQ ID NO:7 Homo sapiens MGAT1 polynucleotide (AF384163)

SEQ ID NO:8 Mus musculus MGAT1 polynucleotide (AF384162)

SEQ ID NO:9 Pan troglodytes MGAT1 polynucleotide transcript variant(XM_001166055)

SEQ ID NO:10 Pan troglodytes MGAT1 polynucleotide transcript variant 2(XM_0526044.2)

SEQ ID NO:11 Canis familiaris MGAT1 polynucleotide (XM_545667.2)

SEQ ID NO:12 Bos taurus MGAT1 polynucleotide (NM_001001153.2)

SEQ ID NO:13 Rattus norvegicus MGAT1 polynucleotide (NM_001108803.1)

SEQ ID NO:14 Danio rerio MGAT1 polynucleotide (NM_001122623.1)

SEQ ID NO:15 Caenorhabditis elegans MGAT1 polynucleotide (NM_073012.4)

SEQ ID NO:16 Caenorhabditis elegans MGAT1 polynucleotide (NM_182380.5)

SEQ ID NO:17 Caenorhabditis elegans MGAT1 polynucleotide (NM_065258.3)

SEQ ID NO:18 Caenorhabditis elegans MGAT1 polynucleotide (NM_075068.3)

SEQ ID NO:19 Caenorhabditis elegans MGAT1 polynucleotide (NM_072248.3)

SEQ ID NO:20 Kluyveromyces lactis MGAT1 polynucleotide (XM_455588.1)

SEQ ID NO:21 Ashbya gossypii MGAT1 polynucleotide (NM_208895.1)

SEQ ID NO:22 Magnaporthe oryzae MGAT1 polynucleotide (XM_368741.1)

SEQ ID NO:23 Ciona intestinalis MGAT1 polynucleotide (XM_002120843.1)

SEQ ID NO:24 Homo sapiens MGAT2 polynucleotide (AY157608)

SEQ ID NO:25 Mus musculus MGAT2 polynucleotide (AY157609)

SEQ ID NO:26 Pan troglodytes MGAT2 polynucleotide (XM_522112.2)

SEQ ID NO:27 Canis familiaris MGAT2 polynucleotide (XM_542304.1)

SEQ ID NO:28 Bos taurus MGAT2 polynucleotide (NM_001099136.1)

SEQ ID NO:29 Rattus norvegicus MGAT2 polynucleotide (NM_001109436.2)

SEQ ID NO:30 Gallus gallus MGAT2 polynucleotide (XM_424082.2)

SEQ ID NO:31 Danio rerio MGAT2 polynucleotide (NM_001006083.1)

SEQ ID NO:32 Drosophila melanogaster MGAT2 polynucleotide (NM_136474.2)

SEQ ID NO:33 Drosophila melanogaster MGAT2 polynucleotide (NM_136473.2)

SEQ ID NO:34 Drosophila melanogaster MGAT2 polynucleotide (NM_136475.2)

SEQ ID NO:35 Anopheles gambiae MGAT2 polynucleotide (XM_001688709.1)

SEQ ID NO:36 Anopheles gambiae MGAT2 polynucleotide (XM_315985)

SEQ ID NO:37 Tribolium castaneum MGAT2 polynucleotide (XM_970053.1)

SEQ ID NO:38 Homo sapiens MGAT3 polynucleotide (AY229854)

SEQ ID NO:39 Pan troglodytes MGAT3 polynucleotide transcript variant 1(XM_001154107.1)

SEQ ID NO:40 Pan troglodytes MGAT3 polynucleotide transcript variant 2(XM_001154171.1)

SEQ ID NO:41 Pan troglodytes MGAT3 polynucleotide transcript variant 3(XM_527842.2)

SEQ ID NO:42 Canis familiaris MGAT3 polynucleotide (XM_845212.1)

SEQ ID NO:43 Bos taurus MGAT3 polynucleotide (XM_870406.4)

SEQ ID NO:44 Danio rerio MGAT3 polynucleotide (XM_688413.4)

SEQ ID NO:45 Homo sapiens MGAT1 polypeptide (AAK84178.1)

SEQ ID NO:46 Mus musculus MGAT1 polypeptide (AAK84177.1)

SEQ ID NO:47 Pan troglodytes MGAT1 polypeptide isoform 1(XP_001166055.1)

SEQ ID NO:48 Pan troglodytes MGAT1 polypeptide isoform 2 (XP_526044.2)

SEQ ID NO:49 Canis familiaris MGAT1 polypeptide (XP_545667.2)

SEQ ID NO:50 Bos taurus MGAT1 polypeptide (NP_001001153.1)

SEQ ID NO:51 Rattus norvegicus MGAT1 polypeptide (NP_001102273.1)

SEQ ID NO:52 Danio rerio MGAT1 polypeptide (NP_001116095.1)

SEQ ID NO:53 Caenorhabditis elegans MGAT1 polypeptide (NP_505413.1)

SEQ ID NO:54 Caenorhabditis elegans MGAT1 polypeptide (NP_872180.1)

SEQ ID NO:55 Caenorhabditis elegans MGAT1 polypeptide (NP_497659.1)

SEQ ID NO:56 Caenorhabditis elegans MGAT1 polypeptide (NP_507469.1)

SEQ ID NO:57 Caenorhabditis elegans MGAT1 polypeptide (NP_504649.1)

SEQ ID NO:58 Kluyveromyces lactis MGAT1 polypeptide (XP_455588.1)

SEQ ID NO:59 Ashbya gossypii MGAT1 polypeptide (NP_983542.1)

SEQ ID NO:60 Magnaporthe oryzae MGAT1 polypeptide (XP_368741.1)

SEQ ID NO:61 Ciona intestinalis MGAT1 polypeptide (XP_002120879)

SEQ ID NO:62 Homo sapiens MGAT2 polypeptide (AA023672.1)

SEQ ID NO:63 Mus musculus MGAT2 polypeptide (AA023673.1)

SEQ ID NO:64 Pan troglodytes MGAT2 polypeptide (XP_522112.2)

SEQ ID NO:65 Canis familiaris MGAT2 polypeptide (XP_542304.1)

SEQ ID NO:66 Bos taurus MGAT2 polypeptide (NP_001092606.1)

SEQ ID NO:67 Rattus norvegicus MGAT2 polypeptide (NP_001102906.2)

SEQ ID NO:68 Gallus gallus MGAT2 polypeptide (XP_424082.2)

SEQ ID NO:69 Danio rerio MGAT2 polypeptide (NP_001006083.1)

SEQ ID NO:70 Drosophila melanogaster MGAT2 polypeptide (NP_610318.1)

SEQ ID NO:71 Drosophila melanogaster MGAT2 polypeptide (NP_610317.1)

SEQ ID NO:72 Drosophila melanogaster MGAT2 polypeptide (NP_610319.2)

SEQ ID NO:73 Anopheles gambiae MGAT2 polypeptide (XP_001688761)

SEQ ID NO:74 Anopheles gambiae MGAT2 polypeptide (XP_315985.3)

SEQ ID NO:75 Tribolium castaneum MGAT2 polypeptide (XP_975146)

SEQ ID NO:76 Homo sapiens MGAT3 polypeptide (AA063579.1)

SEQ ID NO:77 Pan troglodytes MGAT3 polypeptide isoform 1(XP_001154107.1)

SEQ ID NO:78 Pan troglodytes MGAT3 polypeptide isoform 2(XP_001154171.1)

SEQ ID NO:79 Pan troglodytes MGAT3 isoform 3 (XP_527842.2)

SEQ ID NO:80 Canis familiaris MGAT3 polypeptide (XP_850305.1)

SEQ ID NO:81 Bos taurus MGAT3 polypeptide (XP_875499.3)

SEQ ID NO:82 Danio rerio MGAT3 polypeptide (XP_693505.1)

SEQ ID NO:83 Arabidopsis thaliana DGAT1 polypeptide (CAB44774.1)

SEQ ID NO:84 Arabidopsis thaliana GPAT4 polynucleotide (NM_100043.4)

SEQ ID NO:85 Arabidopsis thaliana GPAT6 polynucleotide (NM_129367.3)

SEQ ID NO:86 Arabidopsis thaliana GPAT polynucleotide (AF195115.1)

SEQ ID NO:87 Arabidopsis thaliana GPAT polynucleotide (AY062466.1)

SEQ ID NO:88 Oryza sativa GPAT polynucleotide (AC118133.4)

SEQ ID NO:89 Picea sitchensis GPAT polynucleotide (EF086095.1)

SEQ ID NO:90 Zea mays GPAT polynucleotide (BT067649.1)

SEQ ID NO:91 Arabidopsis thaliana GPAT polynucleotide (AK228870.1)

SEQ ID NO:92 Oryza sativa GPAT polynucleotide (AK241033.1)

SEQ ID NO:93 Oryza sativa GPAT polynucleotide (CM000127.1)

SEQ ID NO:94 Oryza sativa GPAT polynucleotide (CM000130.1)

SEQ ID NO:95 Oryza sativa GPAT polynucleotide (CM000139.1)

SEQ ID NO:96 Oryza sativa GPAT polynucleotide (CM000126.1)

SEQ ID NO:97 Oryza sativa GPAT polynucleotide (CM000128.1)

SEQ ID NO:98 Oryza sativa GPAT polynucleotide (CM000140.1)

SEQ ID NO:99 Selaginella moellendorffii GPAT polynucleotide (GL377667.1)

SEQ ID NO:100 Selaginella moellendorffii GPAT polynucleotide(GL377667.1)

SEQ ID NO:101 Selaginella moellendorffii GPAT polynucleotide(GL377648.1)

SEQ ID NO:102 Selaginella moellendorffii GPAT polynucleotide(GL377622.1)

SEQ ID NO:103 Selaginella moellendorffii GPAT polynucleotide(GL377590.1)

SEQ ID NO:104 Selaginella moellendorffii GPAT polynucleotide(GL377576.1)

SEQ ID NO:105 Selaginella moellendorffii GPAT polynucleotide(GL377576.1)

SEQ ID NO:106 Oryza sativa GPAT polynucleotide (NM_001051374.2)

SEQ ID NO:107 Oryza sativa GPAT polynucleotide (NM_001052203.1)

SEQ ID NO:108: Zea mays GPAT8 polynucleotide (NM_001153970.1)

SEQ ID NO:109: Zea mays GPAT polynucleotide (NM_001155835.1)

SEQ ID NO:110: Zea mays GPAT polynucleotide (NM_001174880.1)

SEQ ID NO:111 Brassica napus GPAT4 polynucleotide (JQ666202.1)

SEQ ID NO:112 Arabidopsis thaliana GPAT8 polynucleotide (NM_116264.5)

SEQ ID NO:113 Physcomitrella patens GPAT polynucleotide (XM_001764949.1)

SEQ ID NO:114 Physcomitrella patens GPAT polynucleotide (XM_001769619.1)

SEQ ID NO:115 Physcomitrella patens GPAT polynucleotide (XM_001769672.1)

SEQ ID NO:116 Physcomitrella patens GPAT polynucleotide (XM_001771134.1)

SEQ ID NO:117 Physcomitrella patens GPAT polynucleotide (XM_001780481.1)

SEQ ID NO:118 Vitis vinifera GPAT polynucleotide (XM_002268477.1)

SEQ ID NO:119 Vitis vinifera GPAT polynucleotide (XM_002275312.1)

SEQ ID NO:120 Vitis vinifera GPAT polynucleotide (XM_002275996.1)

SEQ ID NO:121 Vitis vinifera GPAT polynucleotide (XM_002279055.1)

SEQ ID NO:122 Populus trichocarpa GPAT polynucleotide (XM_002309088.1)

SEQ ID NO:123 Populus trichocarpa GPAT polynucleotide (XM_002309240.1)

SEQ ID NO:124 Populus trichocarpa GPAT polynucleotide (XM_002322716.1)

SEQ ID NO:125 Populus trichocarpa GPAT polynucleotide (XM_002323527.1)

SEQ ID NO:126 Sorghum bicolor GPAT polynucleotide (XM_002439842.1)

SEQ ID NO:127 Sorghum bicolor GPAT polynucleotide (XM_002458741.1)

SEQ ID NO:128 Sorghum bicolor GPAT polynucleotide (XM_002463871.1)

SEQ ID NO:129 Sorghum bicolor GPAT polynucleotide (XM_002464585.1)

SEQ ID NO:130 Ricinus communis GPAT polynucleotide (XM_002511827.1)

SEQ ID NO:131 Ricinus communis GPAT polynucleotide (XM_002517392.1)

SEQ ID NO:132 Ricinus communis GPAT polynucleotide (XM_002520125.1)

SEQ ID NO:133 Arabidopsis lyrata GPAT polynucleotide (XM_002872909.1)

SEQ ID NO:134 Arabidopsis lyrata GPAT6 polynucleotide (XM_002881518.1)

SEQ ID NO 135 Vernicia fordii putative GPAT8 polynucleotide (FJ479753.1)

SEQ ID NO 136 Oryza sativa GPAT polynucleotide (NM_001057724.1)

SEQ ID NO:137 Brassica napus GPAT4 polynucleotide (JQ666203.1) SEQ ID NO138 Populus trichocarpa GPAT polynucleotide (XM_002320102.1)

SEQ ID NO:139 Sorghum bicolor GPAT polynucleotide (XM_002451332.1)

SEQ ID NO:140 Ricinus communis GPAT polynucleotide (XM_002531304.1)

SEQ ID NO:141 Arabidopsis lyrata GPAT4 polynucleotide (XM_002889315.1)

SEQ ID NO:142 Arabidopsis thaliana GPAT1 polynucleotide (NM_100531.2)

SEQ ID NO 143 Arabidopsis thaliana GPAT3 polynucleotide (NM_116426.2)

SEQ ID NO:144 Arabidopsis thaliana GPAT4 polypeptide (NP_171667.1)

SEQ ID NO:145 Arabidopsis thaliana GPAT6 polypeptide (NP_181346.1)

SEQ ID NO:146 Arabidopsis thaliana GPAT polypeptide (AAF02784.1)

SEQ ID NO:147 Arabidopsis thaliana GPAT polypeptide (AAL32544.1)

SEQ ID NO:148 Oryza sativa GPAT polypeptide (AAP03413.1)

SEQ ID NO:149 Picea sitchensis GPAT polypeptide (ABK25381.1)

SEQ ID NO:150 Zea mays GPAT polypeptide (ACN34546.1)

SEQ NO ID:151 Arabidopsis thaliana GPAT polypeptide (BAF00762.1)

SEQ ID NO:152 Oryza sativa GPAT polypeptide (BAH00933.1)

SEQ ID NO:153 Oryza sativa GPAT polypeptide (EAY84189.1)

SEQ ID NO:154 Oryza sativa GPAT polypeptide (EAY98245.1)

SEQ ID NO:155 Oryza sativa GPAT polypeptide (EAZ21484.1)

SEQ ID NO:156 Oryza sativa GPAT polypeptide (EEC71826.1)

SEQ ID NO:157 Oryza sativa GPAT polypeptide (EEC76137.1)

SEQ ID NO:158 Oryza sativa GPAT polypeptide (EEE59882.1)

SEQ ID NO:159 Selaginella moellendorffii GPAT polypeptide (EFJ08963.1)

SEQ ID NO:160 Selaginella moellendorffii GPAT polypeptide (EFJ08964.1)

SEQ ID NO:161 Selaginella moellendorffii GPAT polypeptide (EFJ11200.1)

SEQ ID NO:162 Selaginella moellendorffii GPAT polypeptide (EFJ15664.1)

SEQ ID NO:163 Selaginella moellendorffii GPAT polypeptide (EFJ24086.1)

SEQ ID NO:164 Selaginella moellendorffii GPAT polypeptide (EFJ29816.1)

SEQ ID NO:165 Selaginella moellendorffii GPAT polypeptide (EFJ29817.1)

SEQ ID NO:166 Oryza sativa GPAT polypeptide (NP_001044839.1)

SEQ ID NO:167 Oryza sativa GPAT polypeptide (NP_001045668.1)

SEQ ID NO:168 Zea mays GPAT 8 polypeptide (NP_001147442.1)

SEQ ID NO:169 Zea mays GPAT polypeptide (NP_001149307.1)

SEQ ID NO:170 Zea mays protein GPAT polypeptide (NP_001168351.1)

SEQ ID NO:171 Brassica napus GPAT4 polypeptide (AFH02724.1)

SEQ ID NO:172 Arabidopsis thaliana GPAT8 polypeptide (NP_191950.2)

SEQ ID NO:173 Physcomitrella patens GPAT polypeptide (XP_001765001.1)

SEQ ID NO:174 Physcomitrella patens GPAT polypeptide (XP_001769671.1)

SEQ ID NO:175 Physcomitrella patens GPAT polypeptide (XP_001769724.1)

SEQ ID NO:176 Physcomitrella patens GPAT polypeptide (XP_001771186.1)

SEQ ID NO:177 Physcomitrella patens GPAT polypeptide (XP_001780533.1)

SEQ ID NO:178 Vitis vinifera GPAT polypeptide (XP_002268513.1)

SEQ ID NO:179 Vitis vinifera GPAT polypeptide (XP_002275348.1)

SEQ ID NO:180 Vitis vinifera GPAT polypeptide (XP_002276032.1)

SEQ ID NO:181 Vitis vinifera GPAT polypeptide (XP_002279091.1)

SEQ ID NO:182 Populus trichocarpa GPAT polypeptide (XP_002309124.1)

SEQ ID NO:183 Populus trichocarpa GPAT polypeptide (XP_002309276.1)

SEQ ID NO:184 Populus trichocarpa GPAT polypeptide (XP_002322752.1)

SEQ ID NO:185 Populus trichocarpa GPAT polypeptide (XP_002323563.1)

SEQ ID NO:186 Sorghum bicolor GPAT polypeptide (XP_002439887.1)

SEQ ID NO:187 Sorghum bicolor GPAT polypeptide (XP_002458786.1)

SEQ ID NO:188 Sorghum bicolor GPAT polypeptide (XP_002463916.1)

SEQ ID NO:189 Sorghum bicolor GPAT polypeptide (XP_002464630.1)

SEQ ID NO:190 Ricinus communis GPAT polypeptide (XP_002511873.1)

SEQ ID NO:191 Ricinus communis GPAT polypeptide (XP_002517438.1)

SEQ ID NO:192 Ricinus communis GPAT polypeptide (XP_002520171.1)

SEQ ID NO:193 Arabidopsis lyrata GPAT polypeptide (XP_002872955.1)

SEQ ID NO:194 Arabidopsis lyrata GPAT6 polypeptide (XP_002881564.1)

SEQ ID NO:195 Vernicia fordii GPAT polypeptide (ACT32032.1)

SEQ ID NO:196 Oryza sativa GPAT polypeptide (NP_001051189.1)

SEQ ID NO:197 Brassica napus GPAT4 polypeptide (AFH02725.1)

SEQ ID NO:198 Populus trichocarpa GPAT polypeptide (XP_002320138.1)

SEQ ID NO:199 Sorghum bicolor GPAT polypeptide (XP_002451377.1)

SEQ ID NO:200 Ricinus communis GPAT polypeptide (XP_002531350.1)

SEQ ID NO:201 Arabidopsis lyrata GPAT4 polypeptide (XP_002889361.1)

SEQ ID NO:202 Arabidopsis thaliana GPAT1 polypeptide (NP_563768.1)

SEQ ID NO:203 Arabidopsis thaliana GPAT3 polypeptide (NP_192104.1)

SEQ ID NO:204 Arabidopsis thaliana DGAT2 polynucleotide (NM_115011.3)

SEQ ID NO:205 Ricinus communis DGAT2 polynucleotide (AY916129.1)

SEQ ID NO:206 Vernicia fordii DGAT2 polynucleotide (DQ356682.1)

SEQ ID NO:207 Mortierella ramanniana DGAT2 polynucleotide (AF391089.1)

SEQ ID NO:208 Homo sapiens DGAT2 polynucleotide (NM_032564.1)

SEQ ID NO:209 Homo sapiens DGAT2 polynucleotide (NM_001013579.2)

SEQ ID NO:210 Bos taurus DGAT2 polynucleotide (NM_205793.2)

SEQ ID NO:211 Mus musculus DGAT2 polynucleotide (AF384160.1)

SEQ ID NO:212 Arabidopsis thaliana DGAT2 polypeptide (NP_566952.1)

SEQ ID NO:213 Ricinus communis DGAT2 polypeptide (AAY16324.1)

SEQ ID NO:214 Vernicia fordii DGAT2 polypeptide (ABC94474.1)

SEQ ID NO:215 Mortierella ramanniana DGAT2 polypeptide (AAK84179.1)

SEQ ID NO:216 Homo sapiens DGAT2 polypeptide (Q96PD7.2)

SEQ ID NO:217 Homo sapiens DGAT2 polypeptide (Q58HT5.1)

SEQ ID NO:218 Bos taurus DGAT2 polypeptide (Q70VZ8.1)

SEQ ID NO:219 Mus musculus DGAT2 polypeptide (AAK84175.1)

SEQ ID NO:220 YFP tripeptide—conserved DGAT2 and/or MGAT1/2 sequencemotif

SEQ ID NO:221 HPHG tetrapeptide—conserved DGAT2 and/or MGAT1/2 sequencemotif

SEQ ID NO:222 EPHS tetrapeptide—conserved plant DGAT2 sequence motif

SEQ ID NO:223 RXGFX(K/R)XAXXXGXXX(L/V)VPXXXFG(E/Q)—long conservedsequence motif of DGAT2 which is part of the putative glycerolphospholipid domain

SEQ ID NO:224 FLXLXXXN—conserved sequence motif of mouse DGAT2 andMGAT1/2 which is a putative neutral lipid binding domain

SEQ ID NO:225 plsC acyltransferase domain (PF01553) of GPAT

SEQ ID NO:226 HAD-like hydrolase (PF12710) superfamily domain of GPAT

SEQ ID NO:227 Phosphoserine phosphatase domain (PF00702). GPAT4-8contain a N-terminal region homologous to this domain

SEQ ID NO:228 Conserved GPAT amino acid sequence GDLVICPEGTTCREP

SEQ ID NO:229 Conserved GPAT/phosphatase amino acid sequence (Motif I)

SEQ ID NO:230 Conserved GPAT/phosphatase amino acid sequence (Motif III)

SEQ ID NO:231 Arabidopsis thaliana WRI1 polynucleotide (NM_202701.2)

SEQ ID NO:232 Arabidopsis thaliana WRI1 polynucleotide (NM_001035780.2)

SEQ ID NO:233 Arabidopsis thaliana WRI1 polynucleotide (NM_115292.4)

SEQ ID NO:234 Arabidopsis lyrata subsp. lyrata polynucleotide(XM_002876205.1)

SEQ ID NO:235 Brassica napus WRI1 polynucleotide (DQ370141.1)

SEQ ID NO:236 Brassica napus WRI1 polynucleotide (HM370542.1)

SEQ ID NO:237 Glycine max WRI1 polynucleotide (XM_003530322.1)

SEQ ID NO:238 Jatropha curcas WRI1 polynucleotide (JF703666.1)

SEQ ID NO:239 Ricinus communis WRI1 polynucleotide (XM_002525259.1)

SEQ ID NO:240 Populus trichocarpa WRI1 polynucleotide (XM_002316423.1)

SEQ ID NO:241 Brachypodium distachyon WRI1 polynucleotide(XM_003578949.1)

SEQ ID NO:242 Hordeum vulgare subsp. vulgare WRI1 polynucleotide(AK355408.1)

SEQ ID NO:243 Sorghum bicolor WRI1 polynucleotide (XM_002450149.1)

SEQ ID NO:244 Zea mays WRI1 polynucleotide (EU960249.1)

SEQ ID NO:245 Brachypodium distachyon WRI1 polynucleotide(XM_003561141.1)

SEQ ID NO:246 Sorghum bicolor WRI1 polynucleotide (XM_002437774.1)

SEQ ID NO:247 Sorghum bicolor WRI1 polynucleotide (XM_002441399.1)

SEQ ID NO:248 Glycine max WRI1 polynucleotide (XM_003530638.1)

SEQ ID NO:249 Glycine max WRI1 polynucleotide (XM_003553155.1)

SEQ ID NO:250 Populus trichocarpa WRI1 polynucleotide (XM_002315758.1)

SEQ ID NO:251 Vitis vinifera WRI1 polynucleotide (XM_002270113.1)

SEQ ID NO:252 Glycine max WRI1 polynucleotide (XM_003533500.1)

SEQ ID NO:253 Glycine max WRI1 polynucleotide (XM_003551675.1)

SEQ ID NO:254 Medicago truncatula WRI1 polynucleotide (XM_003621069.1)

SEQ ID NO:255 Populus trichocarpa WRI1 polynucleotide (XM_002323800.1)

SEQ ID NO:256 Ricinus communis WRI1 polynucleotide (XM_002517428.1)

SEQ ID NO:257 Brachypodium distachyon WRI1 polynucleotide(XM_003572188.1)

SEQ ID NO:258 Sorghum bicolor WRI1 polynucleotide (XM_002444384.1)

SEQ ID NO:259 Zea mays WRI1 polynucleotide (NM_001176888.1)

SEQ ID NO:260 Arabidopsis lyrata subsp. lyrata WRI1 polynucleotide(XM_002889219.1)

SEQ ID NO:261 Arabidopsis thaliana WRI1 polynucleotide (NM_106619.3)

SEQ ID NO:262 Arabidopsis lyrata subsp. lyrata WRI1 polynucleotide(XM_002890099.1)

SEQ ID NO:263 Thellungiella halophila WRI1 polynucleotide (AK352786.1)

SEQ ID NO:264 Arabidopsis thaliana WRI1 polynucleotide (NM_101474.2)

SEQ ID NO:265 Glycine max WRI1 polynucleotide (XM_003530302.1)

SEQ ID NO:266 Brachypodium distachyon WRI1 polynucleotide(XM_003578094.1)

SEQ ID NO:267 Sorghum bicolor WRI1 polynucleotide (XM_002460191.1)

SEQ ID NO:268 Zea mays WRI1 polynucleotide (NM_001152866.1)

SEQ ID NO:269 Glycine max WRI1 polynucleotide (XM_003519119.1)

SEQ ID NO:270 Glycine max WRI1 polynucleotide (XM_003550628.1)

SEQ ID NO:271 Medicago truncatula WRI1 polynucleotide (XM_003610213.1)

SEQ ID NO:272 Glycine max WRI1 polynucleotide (XM_003523982.1)

SEQ ID NO:273 Glycine max WRI1 polynucleotide (XM_003525901.1)

SEQ ID NO:274 Populus trichocarpa WRI1 polynucleotide (NM_002325075.1)

SEQ ID NO:275 Vitis vinifera WRI1 polynucleotide (XM_002273010.2)

SEQ ID NO:276 Populus trichocarpa WRI1 polynucleotide (XM_002303830.1)

SEQ ID NO:277 Lupinis angustifolius WRI1 polynucleotide, partialsequence (NA-080818_Plate14f06.b1)

SEQ ID NO:278 Lupinis angustifolius WRI1 polynucleotide

SEQ ID NO:279 Arabidopsis thaliana WRI1 polypeptide (A8MS57)

SEQ ID NO:280 Arabidopsis thaliana WRI1 polypeptide (Q6X5Y6)

SEQ ID NO:281 Arabidopsis lyrata subsp. lyrata WRI1 polypeptide(XP_002876251.1)

SEQ ID NO:282 Brassica napus WRI1 polypeptide (ABD16282.1)

SEQ ID NO:283 Brassica napus WRI1 polypeptide (ADO16346.1)

SEQ ID NO:284 Glycine max WRI1 polypeptide (XP_003530370.1)

SEQ ID NO:285 Jatropha curcas WRI1 polypeptide (AEO22131.1)

SEQ ID NO:286 Ricinus communis WRI1 polypeptide (XP_002525305.1)

SEQ ID NO:287 Populus trichocarpa WRI1 polypeptide (XP_002316459.1)

SEQ ID NO:288 Vitis vinifera WRI1 polypeptide (CBI29147.3)

SEQ ID NO:289 Brachypodium distachyon WRI1 polypeptide (XP_003578997.1)

SEQ ID NO:290 Hordeum vulgare subsp. vulgare WRI1 polypeptide(BAJ86627.1)

SEQ ID NO:291 Oryza sativa WRI1 polypeptide (EAY79792.1)

SEQ ID NO:292 Sorghum bicolor WRI1 polypeptide (XP_002450194.1)

SEQ ID NO:293 Zea mays WRI1 polypeptide (ACG32367.1)

SEQ ID NO:294 Brachypodium distachyon WRI1 polypeptide (XP_003561189.1)

SEQ ID NO:295 Brachypodium sylvaticum WRI1 polypeptide (ABL85061.1)

SEQ ID NO:296 Oryza sativa WRI1 polypeptide (BAD68417.1)

SEQ ID NO:297 Sorghum bicolor WRI1 polypeptide (XP_002437819.1)

SEQ ID NO:298 Sorghum bicolor WRI1 polypeptide (XP_002441444.1)

SEQ ID NO:299 Glycine max WRI1 polypeptide (XP_003530686.1)

SEQ ID NO:300 Glycine max WRI1 polypeptide (XP_003553203.1)

SEQ ID NO:301 Populus trichocarpa WRI1 polypeptide (XP_002315794.1)

SEQ ID NO:302 Vitis vinifera WRI1 polypeptide (XP_002270149.1)

SEQ ID NO:303 Glycine max WRI1 polypeptide (XP_003533548.1)

SEQ ID NO:304 Glycine max WRI1 polypeptide (XP_003551723.1)

SEQ ID NO:305 Medicago truncatula WRI1 polypeptide (XP_003621117.1)

SEQ ID NO:306 Populus trichocarpa WRI1 polypeptide (XP_002323836.1)

SEQ ID NO:307 Ricinus communis WRI1 polypeptide (XP_002517474.1)

SEQ ID NO:308 Vitis vinifera WRI1 polypeptide (CAN79925.1)

SEQ ID NO:309 Brachypodium distachyon WRI1 polypeptide (XP_003572236.1)

SEQ ID NO:310 Oryza sativa WRI1 polypeptide (BAD10030.1)

SEQ ID NO:311 Sorghum bicolor WRI1 polypeptide (XP_002444429.1)

SEQ ID NO:312 Zea mays WRI1 polypeptide (NP_001170359.1)

SEQ ID NO:313 Arabidopsis lyrata subsp. lyrata WRI1 polypeptide(XP_002889265.1)

SEQ ID NO:314 Arabidopsis thaliana WRI1 polypeptide (AAF68121.1)

SEQ ID NO:315 Arabidopsis thaliana WRI1 polypeptide (NP_178088.2)

SEQ ID NO:316 Arabidopsis lyrata subsp. lyrata WRI1 polypeptide(XP_002890145.1)

SEQ ID NO:317 Thellungiella halophila WRI1 polypeptide (BAJ33872.1)

SEQ ID NO:318 Arabidopsis thaliana WRI1 polypeptide (NP_563990.1)

SEQ ID NO:319 Glycine max WRI1 polypeptide (XP_003530350.1)

SEQ ID NO:320 Brachypodium distachyon WRI1 polypeptide (XP_003578142.1)

SEQ ID NO:321 Oryza sativa WRI1 polypeptide (EAZ09147.1)

SEQ ID NO:322 Sorghum bicolor WRI1 polypeptide (XP_002460236.1)

SEQ ID NO:323 Zea mays WRI1 polypeptide (NP_001146338.1)

SEQ ID NO:324 Glycine max WRI1 polypeptide (XP_003519167.1)

SEQ ID NO:325 Glycine max WRI1 polypeptide (XP_003550676.1)

SEQ ID NO:326 Medicago truncatula WRI1 polypeptide (XP_003610261.1)

SEQ ID NO:327 Glycine max WRI1 polypeptide (XP_003524030.1)

SEQ ID NO:328 Glycine max WRI1 polypeptide (XP_003525949.1)

SEQ ID NO:329 Populus trichocarpa WRI1 polypeptide (XP_002325111.1)

SEQ ID NO:330 Vitis vinifera WRI1 polypeptide (CBI36586.3)

SEQ ID NO:331 Vitis vinifera WRI1 polypeptide (XP_002273046.2)

SEQ ID NO:332 Populus trichocarpa WRI1 polypeptide (XP_002303866.1)

SEQ ID NO:333 Vitis vinifera WRI1 polypeptide (CBI25261.3)

SEQ ID NO:334 Sorbi-WRL1

SEQ ID NO: 335 Lupan-WRL1

SEQ ID NO:336 Ricco-WRL1

SEQ ID NO:337 Lupin angustifolius WRI1 polypeptide

SEQ ID NO:338 Aspergillus fumigatus DGAT polynucleotide (XM_750079.1)

SEQ ID NO:339 Ricinus communis DGAT polynucleotide (AY366496.1)

SEQ ID NO:340 Vernicia fordii DGAT1 polynucleotide (DQ356680.1)

SEQ ID NO:341 Vernonia galamensis DGAT1 polynucleotide (EF653276.1)

SEQ ID NO:342 Vernonia galamensis DGAT1 polynucleotide (EF653277.1)

SEQ ID NO:343 Euonymus alatus DGAT1 polynucleotide (AY751297.1)

SEQ ID NO:344 Caenorhabditis elegans DGAT1 polynucleotide (AF221132.1)

SEQ ID NO:345 Rattus norvegicus DGAT1 polynucleotide (NM_053437.1)

SEQ ID NO:346 Homo sapiens DGAT1 polynucleotide (NM_012079.4)

SEQ ID NO:347 Aspergillus fumigatus DGAT1 polypeptide (XP_755172.1)

SEQ ID NO:348 Ricinus communis DGAT1 polypeptide (AAR11479.1)

SEQ ID NO:349 Vernicia fordii DGAT1 polypeptide (ABC94472.1)

SEQ ID NO:350 Vernonia galamensis DGAT1 polypeptide (ABV21945.1)

SEQ ID NO:351 Vernonia galamensis DGAT1 polypeptide (ABV21946.1)

SEQ ID NO:352 Euonymus alatus DGAT1 polypeptide (AAV31083.1)

SEQ ID NO:353 Caenorhabditis elegans DGAT1 polypeptide (AAF82410.1)

SEQ ID NO:354 Rattus norvegicus DGAT1 polypeptide (NP_445889.1)

SEQ ID NO:355 Homo sapiens DGAT1 polypeptide (NP_036211.2)

SEQ ID NO:356 WRI1 motif (R G V T/S R H R W T G R)

SEQ ID NO:357 WRI1 motif (F/Y E A H L W D K)

SEQ ID NO:358 WRI1 motif (D L A A L K Y W G)

SEQ ID NO:359 WRI1 motif (S X G F S/A R G X)

SEQ ID NO:360 WRI1 motif (H H H/Q N G R/K W E A R I G R/K V)

SEQ ID NO:361 WRI1 motif (Q E E A A A X Y D)

SEQ ID NO:362 Brassica napus oleosin polypeptide (CAA57545.1)

SEQ ID NO:363 Brassica napus oleosin S1-1 polypeptide (ACG69504.1)

SEQ ID NO:364 Brassica napus oleosin S2-1 polypeptide (ACG69503.1)

SEQ ID NO:365 Brassica napus oleosin S3-1 polypeptide (ACG69513.1)

SEQ ID NO:366 Brassica napus oleosin S4-1 polypeptide (ACG69507.1)

SEQ ID NO:367 Brassica napus oleosin S5-1 polypeptide (ACG69511.1)

SEQ ID NO:368 Arachis hypogaea oleosin 1 polypeptide (AAZ20276.1)

SEQ ID NO:369 Arachis hypogaea oleosin 2 polypeptide (AAU21500.1)

SEQ ID NO:370 Arachis hypogaea oleosin 3 polypeptide (AAU21501.1)

SEQ ID NO:371 Arachis hypogaea oleosin 5 polypeptide (ABC96763.1)

SEQ ID NO:372 Ricinus communis oleosin 1 polypeptide (EEF40948.1)

SEQ ID NO:373 Ricinus communis oleosin 2 polypeptide (EEF51616.1)

SEQ ID NO:374 Glycine max oleosin isoform a polypeptide (P29530.2)

SEQ ID NO:375 Glycine max oleosin isoform b polypeptide (P29531.1)

SEQ ID NO:376 Linum usitatissimum oleosin low molecular weight isoformpolypeptide (ABB01622.1)

SEQ ID NO:377 amino acid sequence of Linum usitatissimum oleosin highmolecular weight isoform polypeptide (ABB01624.1)

SEQ ID NO:378 Helianthus annuus oleosin polypeptide (CAA44224.1)

SEQ ID NO:379 Zea mays oleosin polypeptide (NP_001105338.1)

SEQ ID NO:380 Brassica napus steroleosin polypeptide (ABM30178.1)

SEQ ID NO:381 Brassica napus steroleosin SLO1-1 polypeptide (ACG69522.1)

SEQ ID NO:382 Brassica napus steroleosin SLO2-1 polypeptide (ACG69525.1)

SEQ ID NO:383 Sesamum indicum steroleosin polypeptide (AAL13315.1)

SEQ ID NO:384 Zea mays steroleosin polypeptide (NP_001152614.1)

SEQ ID NO:385 Brassica napus caleosin CLO-1 polypeptide (ACG69529.1)

SEQ ID NO:386 Brassica napus caleosin CLO-3 polypeptide (ACG69527.1)

SEQ ID NO:387 Sesamum indicum caleosin polypeptide (AAF13743.1)

SEQ ID NO:388 Zea mays caleosin polypeptide (NP_001151906.1)

SEQ ID NO:389 Brassica napus oleosin polynucleotide (X82020.1)

SEQ ID NO:390 Brassica napus oleosin S1-1 polynucleotide (EU678256.1)

SEQ ID NO:391 Brassica napus oleosin S2-1 polynucleotide (EU678255.1)

SEQ ID NO:392 Brassica napus oleosin S3-1 polynucleotide (EU678265.1)

SEQ ID NO:393 Brassica napus oleosin S4-1 polynucleotide (EU678259.1)

SEQ ID NO:394 Brassica napus oleosin S5-1 polynucleotide (EU678263.1)

SEQ ID NO:395 Arachis hypogaea oleosin 1 polynucleotide (DQ097716.1)

SEQ ID NO:396 Arachis hypogaea oleosin 2 polynucleotide (AY722695.1)

SEQ ID NO:397 Arachis hypogaea oleosin 3 polynucleotide (AY722696.1)

SEQ ID NO:398 Arachis hypogaea oleosin 5 polynucleotide (DQ368496.1)

SEQ ID NO:399 Helianthus annuus oleosin polynucleotide (X62352.1)

SEQ ID NO:400 Zea mays oleosin polynucleotide (NM_001111868.1)

SEQ ID NO:401 Brassica napus steroleosin polynucleotide (EF143915.1)

SEQ ID NO:402 Brassica napus steroleosin SLO1-1 polynucleotide(EU678274.1)

SEQ ID NO:403 Brassica napus steroleosin SLO2-1 polynucleotide(EU678277.1)

SEQ ID NO:404 Zea mays steroleosin polynucleotide (NM_001159142.1)

SEQ ID NO:405 Brassica napus caleosin CLO-1 polynucleotide (EU678281.1)

SEQ ID NO:406 Brassica napus caleosin CLO-3 polynucleotide (EU678279.1)

SEQ ID NO:407 Sesamum indicum caleosin polynucleotide (AF109921.1)

SEQ ID NO:408 Zea mays caleosin polynucleotide (NM_001158434.1)

SEQ ID NO:409 pJP3502 entire vector sequence (three-gene)

SEQ ID NO:410 pJP3503 entire vector sequence (four-gene)

SEQ ID NO:411 pJP3502 TDNA (inserted into genome) sequence

SEQ ID NO:412 pJP3503 TDNA (inserted into genome) sequence

SEQ ID NO:413 pJP3507 vector sequence

SEQ ID NO:414 Linker sequence

SEQ ID NO:415 Soybean Synergy

SEQ ID NO:416 12ABFJYC_pJP3569_insert

SEQ ID NO:417 Partial N. benthamiana CGI-58 sequence selected for hpRNAisilencing (pTV46)

SEQ ID NO:418 Partial N. tabacum AGPase sequence selected for hpRNAisilencing (pTV35)

SEQ ID NO:419 GXSXG lipase motif

SEQ ID NO:420 HX(4)D acyltransferase motif

SEQ ID NO:421 VX(3)HGF probable lipid binding motif

SEQ ID NO:422 Arabidopsis thaliana CGi58 polynucleotide (NM_118548.1)

SEQ ID NO:423: Brachypodium distachyon CGi58 polynucleotide(XM_003578402.1)

SEQ ID NO:424 Glycine max CGi58 polynucleotide (XM_003523590.1)

SEQ ID NO:425 Zea mays CGi58 polynucleotide (NM_001155541.1)

SEQ ID NO:426 Sorghum bicolor CGi58 polynucleotide (XM_002460493.1)

SEQ ID NO:427 Ricinus communis CGi58 polynucleotide (XM_002510439.1)

SEQ ID NO:428 Medicago truncatula CGi58 polynucleotide (XM_003603685.1)

SEQ ID NO:429 Arabidopsis thaliana CGi58 polypeptide (NP_194147.2)

SEQ ID NO:430 Brachypodium distachyon CGi58 polypeptide (XP_003578450.1)

SEQ ID NO:431 Glycine max CGi58 polypeptide (XP_003523638.1)

SEQ ID NO:432 Zea Mays CGi58 polypeptide (NP_001149013.1)

SEQ ID NO:433 Sorghum bicolor CGi58 polypeptide (XP_002460538.1)

SEQ ID NO:434 Ricinus communis CGi58 polypeptide (XP_002510485.1)

SEQ ID NO:435 Medicago truncatula CGi58 polypeptide (XP_003603733.1)

SEQ ID NO:436 Oryza sativa CGi58 polypeptide (EAZ09782.1)

SEQ ID NO:437 Arabidopsis thaliana LEC2 polynucleotide (NM_102595.2)

SEQ ID NO:438 Medicago truncatula LEC2 polynucleotide (X60387.1)

SEQ ID NO:439 Brassica napus LEC2 polynucleotide (HM370539.1)

SEQ ID NO:440 Arabidopsis thaliana BBM polynucleotide (NM_121749.2)

SEQ ID NO:441 Medicago truncatula BBM polynucleotide (AY899909.1)

SEQ ID NO:442 Arabidopsis thaliana LEC2 polypeptide (NP_564304.1)

SEQ ID NO:443 Medicago truncatula LEC2 polypeptide (CAA42938.1)

SEQ ID NO:444 Brassica napus LEC2 polypeptide (ADO16343.1)

SEQ ID NO:445 Arabidopsis thaliana BBM polypeptide (NP_197245.2)

SEQ ID NO:446 Medicago truncatula BBM polypeptide (AAW82334.1)

SEQ ID NO:447 Inducible Aspergillus niger alcA promoter

SEQ ID NO:448 AlcR inducer that activates the AlcA promoter in thepresence of ethanol

DETAILED DESCRIPTION OF THE INVENTION General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in cell culture,molecular genetics, immunology, immunohistochemistry, protein chemistry,lipid and fatty acid chemistry, biofuel production, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, andimmunological techniques utilized in the present invention are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature in sources such as, J.Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons(1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory Press (1989), T. A. Brown (editor), EssentialMolecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press(1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A PracticalApproach, Volumes 1-4, IRL Press (1995 and 1996), F. M. Ausubel et al.(editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent), Ed Harlow and David Lane (editors) Antibodies: A LaboratoryManual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al.(editors) Current Protocols in Immunology, John Wiley & Sons (includingall updates until present).

Selected Definitions

The term “transgenic non-human organism” refers to, for example, a wholeplant, alga, non-human animal, or an organism suitable for fermentationsuch as a yeast or fungus, comprising an exogenous polynucleotide(transgene) or an exogenous polypeptide. In an embodiment, thetransgenic non-human organism is not an animal or part thereof. In oneembodiment, the transgenic non-human organism is a phototrophic organism(for example, a plant or alga) capable of obtaining energy from sunlightto synthesize organic compounds for nutrition. In another embodiment,the transgenic non-human organism is a photosyntheic bacterium.

The term “exogenous” in the context of a polynucleotide or polypeptiderefers to the polynucleotide or polypeptide when present in a cell whichdoes not naturally comprise the polynucleotide or polypeptide. Such acell is referred to herein as a “recombinant cell” or a “transgeniccell”. In an embodiment, the exogenous polynucleotide or polypeptide isfrom a different genus to the cell comprising the exogenouspolynucleotide or polypeptide. In another embodiment, the exogenouspolynucleotide or polypeptide is from a different species. In oneembodiment the exogenous polynucleotide or polypeptide is expressed in ahost plant or plant cell and the exogenous polynucleotide or polypeptideis from a different species or genus. The exogenous polynucleotide orpolypeptide may be non-naturally occurring, such as for example, asynthetic DNA molecule which has been produced by recombinant DNAmethods. The DNA molecule may, often preferably, include a proteincoding region which has been codon-optimised for expression in the cell,thereby producing a polypeptide which has the same amino acid sequenceas a naturally occurring polypeptide, even though the nucleotidesequence of the protein coding region is non-naturally occurring. Theexogenous polynucleotide may encode, or the exogenous polypeptide maybe: a diacylglycerol acyltransferase (DGAT) such as a DGAT1 or a DGAT2,a glycerol-3-phosphate acyltransferase (GPAT) such as a GPAT which iscapable of synthesising MAG, a Wrinkled 1 (WRI1) transcription factor,an Oleosin, or a silencing suppressor polypeptide. In one embodiment,the exogenous polypeptide is an exogenous MGAT such as an MGAT1 or anMGAT2.

As used herein, the term “extracted lipid” refers to a compositionextracted from a transgenic organism or part thereof which comprises atleast 60% (w/w) lipid.

As used herein, the term “non-polar lipid” refers to fatty acids andderivatives thereof which are soluble in organic solvents but insolublein water. The fatty acids may be free fatty acids and/or in anesterified form. Examples of esterified forms include, but are notlimited to, triacylglycerol (TAG), diacylyglycerol (DAG),monoacylglycerol (MAG). Non-polar lipids also include sterols, sterolesters and wax esters. Non-polar lipids are also known as “neutrallipids”. Non-polar lipid is typically a liquid at room temperature.Preferably, the non-polar lipid predominantly (>50%) comprises fattyacids that are at least 16 carbons in length. More preferably, at least50% of the total fatty acids in the non-polar lipid are C18 fatty acidsfor example, oleic acid. In an embodiment, at least 50%, more preferablyat least 70%, more preferably at least 80%, more preferably at least90%, more preferably at least 91%, more preferably at least 92%, morepreferably at least 93%, more preferably at least 94%, more preferablyat least 95%, more preferably at least 96%, more preferably at least97%, more preferably at least 98%, more preferably at least 99% of thefatty acids in non-polar lipid of the invention can be found as TAG. Thenon-polar lipid may be further purified or treated, for example byhydrolysis with a strong base to release the free fatty acid, or byfractionation, distillation, or the like. Non-polar lipid may be presentin or obtained from plant parts such as seed, leaves or fruit, fromrecombinant cells or from non-human organisms such as yeast. Non-polarlipid of the invention may form part of “seedoil” if it is obtained fromseed. The free and esterified sterol (for example, sitosterol,campesterol, stigmasterol, brassicasterol, Δ5-avenasterol, sitostanol,campestanol, and cholesterol) concentrations in the extracted lipid maybe as described in Phillips et al., 2002. Sterols in plant oils arepresent as free alcohols, esters with fatty acids (esterified sterols),glycosides and acylated glycosides of sterols. Sterol concentrations innaturally occurring vegetable oils (seedoils) ranges up to a maximum ofabout 1100 mg/100 g. Hydrogenated palm oil has one of the lowestconcentrations of naturally occurring vegetable oils at about 60 mg/100g. The recovered or extracted seedoils of the invention preferably havebetween about 100 and about 1000 mg total sterol/100 g of oil. For useas food or feed, it is preferred that sterols are present primarily asfree or esterified forms rather than glycosylated forms. In the seedoilsof the present invention, preferably at least 50% of the sterols in theoils are present as esterified sterols, except for soybean seedoil whichhas about 25% of the sterols esterified. The canola seedoil and rapeseedoil of the invention preferably have between about 500 and about 800 mgtotal sterol/100 g, with sitosterol the main sterol and campesterol thenext most abundant. The corn seedoil of the invention preferably hasbetween about 600 and about 800 mg total sterol/100 g, with sitosterolthe main sterol. The soybean seedoil of the invention preferably hasbetween about 150 and about 350 mg total sterol/100 g, with sitosterolthe main sterol and stigmasterol the next most abundant, and with morefree sterol than esterified sterol. The cottonseed oil of the inventionpreferably has between about 200 and about 350 mg total sterol/100 g,with sitosterol the main sterol. The coconut oil and palm oil of theinvention preferably have between about 50 and about 100 mg totalsterol/100 g, with sitosterol the main sterol. The safflower seedoil ofthe invention preferably has between about 150 and about 250 mg totalsterol/100 g, with sitosterol the main sterol. The peanut seedoil of theinvention preferably has between about 100 and about 200 mg totalsterol/100 g, with sitosterol the main sterol. The sesame seedoil of theinvention preferably has between about 400 and about 600 mg totalsterol/100 g, with sitosterol the main sterol. The sunflower seedoil ofthe invention preferably has between about 200 and 400 mg totalsterol/100 g, with sitosterol the main sterol. Oils obtained fromvegetative plant parts according to the invention preferably have lessthan 200 mg total sterol/100 g, more preferably less than 100 mg totalsterol/100 g, and most preferably less than 50 mg total sterols/100 g,with the majority of the sterols being free sterols.

As used herein, the term “seedoil” refers to a composition obtained fromthe seed/grain of a plant which comprises at least 60% (w/w) lipid, orobtainable from the seed/grain if the seedoil is still present in theseed/grain. That is, seedoil of the invention includes seedoil which ispresent in the seed/grain or portion thereof, as well as seedoil whichhas been extracted from the seed/grain. The seedoil is preferablyextracted seedoil. Seedoil is typically a liquid at room temperature.Preferably, the total fatty acid (TFA) content in the seedoilpredominantly (>50%) comprises fatty acids that are at least 16 carbonsin length. More preferably, at least 50% of the total fatty acids in theseedoil are C18 fatty acids for example, oleic acid. The fatty acids aretypically in an esterified form such as for example, TAG, DAG, acyl-CoAor phospholipid. The fatty acids may be free fatty acids and/or in anesterified form. In an embodiment, at least 50%, more preferably atleast 70%, more preferably at least 80%, more preferably at least 90%,more preferably at least 91%, more preferably at least 92%, morepreferably at least 93%, more preferably at least 94%, more preferablyat least 95%, more preferably at least 96%, more preferably at least97%, more preferably at least 98%, more preferably at least 99% of thefatty acids in seedoil of the invention can be found as TAG. In anembodiment, seedoil of the invention is “substantially purified” or“purified” oil that has been separated from one or more other lipids,nucleic acids, polypeptides, or other contaminating molecules with whichit is associated in the seed or in a crude extract. It is preferred thatthe substantially purified seedoil is at least 60% free, more preferablyat least 75% free, and more preferably, at least 90% free from othercomponents with which it is associated in the seed or extract. Seedoilof the invention may further comprise non-fatty acid molecules such as,but not limited to, sterols. In an embodiment, the seedoil is canola oil(Brassica sp. such as Brassica carinata, Brassica juncea, Brassicanapobrassica, Brassica napus) mustard oil (Brassica juncea), otherBrassica oil (e.g., Brassica napobrassica, Brassica camelina), sunfloweroil (Helianthus sp. such as Helianthus annuus), linseed oil (Linumusitatissimum), soybean oil (Glycine max), safflower oil (Carthamustinctorius), corn oil (Zea mays), tobacco oil (Nicotiana sp. such asNicotiana tabacum or Nicotiana benthamiana), peanut oil (Arachishypogaea), palm oil (Elaeis guineensis), cottonseed oil (Gossypiumhirsutum), coconut oil (Cocos nucifera), avocado oil (Persea americana),olive oil (Olea europaea), cashew oil (Anacardium occidentale),macadamia oil (Macadamia intergrifolia), almond oil (Prunus amygdalus),oat seed oil (Avena sativa), rice oil (Oryza sp. such as Oryza sativaand Oryza glaberrima), Arabidopsis seed oil (Arabidopsis thaliana), oroil from the seed of Acrocomia aculeata (macauba palm), Aracinishypogaea (peanut), Astrocaryum murumuru (murumuru), Astrocaryum vulgare(tucumã), Attalea geraensis (Indaiá-rateiro), Attalea humilis (Americanoil palm), Attalea oleifera (andaiá), Attalea phalerata (uricuri),Attalea speciosa (babassu), Beta vulgaris (sugar beet), Camelina sativa(false flax), Caryocar brasiliense (pequi), Crambe abyssinica(Abyssinian kale), Cucumis melo (melon), Hordeum vulgare (barley),Jatropha curcas (physic nut), Joannesia princeps (arara nut-tree),Licania rigida (oiticica), Lupinus angustifolius (lupin), Mauritiaflexuosa (buriti palm), Maximiliana maripa (inaja palm), Miscanthus sp.such as Miscanthus×giganteus and Miscanthus sinensis, Oenocarpus bacaba(bacaba-do-azeite), Oenocarpus bataua (patauã), Oenocarpus distichus(bacaba-de-leque), Panicum virgatum (switchgrass), Paraqueiba paraensis(mari), Persea amencana (avocado), Pongamia pinnata (Indian beech),Populus trichocarpa, Ricinus communis (castor), Saccharum sp.(sugarcane), Sesamum indicum (sesame), Solanum tuberosum (potato),Sorghum sp. such as Sorghum bicolor, Sorghum vulgare, Theobromagrandiforum (cupuassu), Trifolium sp., Trithrinax brasiliensis(Brazilian needle palm) and Triticum sp. (wheat) such as Triticumaestivum. Seedoil may be extracted from seed/grain by any method knownin the art. This typically involves extraction with nonpolar solventssuch as diethyl ether, petroleum ether, chloroform/methanol or butanolmixtures, generally associated with first crushing of the seeds. Lipidsassociated with the starch in the grain may be extracted withwater-saturated butanol. The seedoil may be “de-gummed” by methods knownin the art to remove polysaccharides or treated in other ways to removecontaminants or improve purity, stability, or colour. The TAGs and otheresters in the seedoil may be hydrolysed to release free fatty acids, orthe seedoil hydrogenated, treated chemically, or enzymatically as knownin the art.

As used herein, the term “fatty acid” refers to a carboxylic acid with along aliphatic tail of at least 8 carbon atoms in length, eithersaturated or unsaturated. Typically, fatty acids have a carbon-carbonbonded chain of at least 12 carbons in length. Most naturally occurringfatty acids have an even number of carbon atoms because theirbiosynthesis involves acetate which has two carbon atoms. The fattyacids may be in a free state (non-esterified) or in an esterified formsuch as part of a TAG, DAG, MAG, acyl-CoA (thio-ester) bound, or othercovalently bound form. When covalently bound in an esterified form, thefatty acid is referred to herein as an “acyl” group. The fatty acid maybe esterified as a phospholipid such as a phosphatidylcholine (PC),phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol,phosphatidylinositol, or diphosphatidylglycerol. Saturated fatty acidsdo not contain any double bonds or other functional groups along thechain. The term “saturated” refers to hydrogen, in that all carbons(apart from the carboxylic acid [—COOH] group) contain as many hydrogensas possible. In other words, the omega (ω) end contains 3 hydrogens(CH3—) and each carbon within the chain contains 2 hydrogens (—CH2—).Unsaturated fatty acids are of similar form to saturated fatty acids,except that one or more alkene functional groups exist along the chain,with each alkene substituting a singly-bonded “—CH2—CH2—” part of thechain with a doubly-bonded “—CH═CH—” portion (that is, a carbon doublebonded to another carbon). The two next carbon atoms in the chain thatare bound to either side of the double bond can occur in a cis or transconfiguration.

As used herein, the terms “polyunsaturated fatty acid” or “PUFA” referto a fatty acid which comprises at least 12 carbon atoms in its carbonchain and at least two alkene groups (carbon-carbon double bonds). ThePUFA content of the vegetative plant part, or the non-human organism orpart thereof of the invention may be increased or decreased depending onthe combination of exogenous polynucleotides expressed in the vegetativeplant part, or non-human organism or part thereof, or seed of theinvention. For example, when an MGAT is expressed the PUFA leveltypically increases, whereas when DGAT1 is expressed alone or incombination with Mall, the PUFA level is typically decreased due to anincrease in the level of oleic acid. Furthermore, if Δ12 desaturaseactivity is reduced, for example by silencing an endogenous Δ12desaturase, PUFA content is unlikely to increase in the absence of anexogenous polynucleotide encoding a different Δ12 desaturase.

“Monoacylglyceride” or “MAG” is glyceride in which the glycerol isesterified with one fatty acid. As used herein, MAG comprises a hydroxylgroup at an sn-1/3 (also referred to herein as sn-1 MAG or 1-MAG or1/3-MAG) or sn-2 position (also referred to herein as 2-MAG), andtherefore MAG does not include phosphorylated molecules such as PA orPC. MAG is thus a component of neutral lipids in a cell.

“Diacylglyceride” or “DAG” is glyceride in which the glycerol isesterified with two fatty acids which may be the same or, preferably,different. As used herein, DAG comprises a hydroxyl group at a sn-1,3 orsn-2 position, and therefore DAG does not include phosphorylatedmolecules such as PA or PC. DAG is thus a component of neutral lipids ina cell. In the Kennedy pathway of DAG synthesis (FIG. 1), the precursorsn-glycerol-3-phosphate (G-3-P) is esterified to two acyl groups, eachcoming from a fatty acid coenzyme A ester, in a first reaction catalysedby a glycerol-3-phosphate acyltransferase (GPAT) at position sn-1 toform LysoPA, followed by a second acylation at position sn-2 catalysedby a lysophosphatidic acid acyltransferase (LPAAT) to form phosphatidicacid (PA). This intermediate is then de-phosphorylated to form DAG. Inan alternative anabolic pathway (FIG. 1), DAG may be formed by theacylation of either sn-1 MAG or preferably sn-2 MAG, catalysed by MGAT.DAG may also be formed from TAG by removal of an acyl group by a lipase,or from PC essentially by removal of a choline headgroup by any of theenzymes CPT, PDCT or PLC (FIG. 1).

“Triacylglyceride” or “TAG” is glyceride in which the glycerol isesterified with three fatty acids which may be the same (e.g. as intri-olein) or, more commonly, different. In the Kennedy pathway of TAGsynthesis, DAG is formed as described above, and then a third acyl groupis esterified to the glycerol backbone by the activity of DGAT.Alternative pathways for formation of TAG include one catalysed by theenzyme PDAT and the MGAT pathway described herein.

As used herein, the term “acyltransferase” refers to a protein which iscapable of transferring an acyl group from acyl-CoA onto a substrate andincludes MGATs, GPATs and DGATs.

As used herein, the term “Wrinkled 1” or “WRI1” or “WRL1” refers to atranscription factor of the AP2/ERWEBP class which regulates theexpression of several enzymes involved in glycolysis and de novo fattyacid biosynthesis. WRI1 has two plant-specific (AP2/EREB) DNA-bindingdomains. WRI1 in at least Arabidopsis also regulates the breakdown ofsucrose via glycolysis thereby regulating the supply of precursors forfatty acid biosynthesis. In other words, it controls the carbon flowfrom the photosynthate to storage lipids. wri1 mutants have wrinkledseed phenotype, due to a defect in the incorporation of sucrose andglucose into TAGs.

Examples of genes which are transcribed by WRI1 include, but are notlimited to, one or more, preferably all, of pyruvate kinase (At5g52920,At3g22960), pyruvate dehydrogenase (PDH) E1alpha subunit (At1g01090),acetyl-CoA carboxylase (ACCase), BCCP2 subunit (At5g15530), enoyl-ACPreductase (At2g05990; EAR), phosphoglycerate mutase (At1g22170),cytosolic fructokinase, and cytosolic phosphoglycerate mutase, sucrosesynthase (SuSy) (see, for example, Liu et al., 2010b; Baud et al., 2007;Ruuska et al., 2002).

WRL1 contains the conserved domain AP2 (cd00018). AP2 is a DNA-bindingdomain found in transcription regulators in plants such as APETALA2 andEREBP (ethylene responsive element binding protein). In EREBPs thedomain specifically binds to the 11 bp GCC box of the ethylene responseelement (ERE), a promotor element essential for ethylene responsiveness.EREBPs and the C-repeat binding factor CBF1, which is involved in stressresponse, contain a single copy of the AP2 domain. APETALA2-likeproteins, which play a role in plant development contain two copies.

Other sequence motifs in WRI1 and its functional homologs include:

(SEQ ID NO: 356) 1. R G V T/S R H R W T G R. (SEQ ID NO: 357) 2.F/Y E A H L W D K. (SEQ ID NO: 358) 3. D L A A L K Y W G.(SEQ ID NO: 359) 4. S X G F S/A R G X. (SEQ ID NO: 360) 5.H H H/Q N G R/K W E A R I G R/K V. (SEQ ID NO: 361) 6.Q E E A A A X Y D.

As used herein, the term “Wrinkled 1” or “WRIT” also includes “Wrinkled1-like” or “WRI1-like” proteins. Examples of WRI1 proteins includeAccession Nos: Q6X5Y6, (Arabidopsis thaliana; SEQ ID NO:280), XP002876251.1 (Arabidopsis lyrata subsp. Lyrata; SEQ ID NO:281),ABD16282.1 (Brassica napus; SEQ ID NO:282), ADO16346.1 (Brassica napus;SEQ ID NO:283), XP_003530370.1 (Glycine max; SEQ ID NO:284), AEO22131.1(Jatropha curcas; SEQ ID NO:285), XP_002525305.1 (Ricinus communis; SEQID NO:286), XP_002316459.1 (Populus trichocarpa; SEQ ID NO:287),CBI29147.3 (Vitis vinifera; SEQ ID NO:288), XP_003578997.1 (Brachypodiumdistachyon; SEQ ID NO:289), BAJ86627.1 (Hordeum vulgare subsp. vulgare;SEQ ID NO:290), EAY79792.1 (Oryza sativa; SEQ ID NO:291), XP_002450194.1(Sorghum bicolor; SEQ ID NO:292), ACG32367.1 (Zea mays; SEQ ID NO:293),XP_003561189.1 (Brachypodium distachyon; SEQ ID NO:294), ABL85061.1(Brachypodium sylvaticum; SEQ ID NO:295), BAD68417.1 (Oryza sativa; SEQID NO:296), XP_002437819.1 (Sorghum bicolor; SEQ ID NO:297),XP_002441444.1 (Sorghum bicolor; SEQ ID NO:298), XP_003530686.1 (Glycinemax; SEQ ID NO:299), XP_003553203.1 (Glycine max; SEQ ID NO:300),XP_002315794.1 (Populus trichocarpa; SEQ ID NO:301), XP_002270149.1(Vitis vinifera; SEQ ID NO:302), XP_003533548.1 (Glycine max; SEQ IDNO:303), XP_003551723.1 (Glycine max; SEQ ID NO:304), XP_003621117.1(Medicago truncatula; SEQ ID NO:305), XP_002323836.1 (Populustrichocarpa; SEQ ID NO:306), XP_002517474.1 (Ricinus communis; SEQ IDNO:307), CAN79925.1 (Vitis vinifera; SEQ ID NO:308), XP_003572236.1(Brachypodium distachyon; SEQ ID NO:309), BAD10030.1 (Oryza sativa; SEQID NO:310), XP_002444429.1 (Sorghum bicolor; SEQ ID NO:311),NP_001170359.1 (Zea mays; SEQ ID NO:312), XP_002889265.1 (Arabidopsislyrata subsp. lyrata; SEQ ID NO:313), AAF68121.1 (Arabidopsis thaliana;SEQ ID NO:314), NP_178088.2 (Arabidopsis thaliana; SEQ ID NO:315),XP_002890145.1 (Arabidopsis lyrata subsp. lyrata; SEQ ID NO:316),BAJ33872.1 (Thellungiella halophila; SEQ ID NO:317), NP_563990.1(Arabidopsis thaliana; SEQ ID NO:318), XP_003530350.1 (Glycine max; SEQID NO:319), XP_003578142.1 (Brachypodium distachyon; SEQ ID NO:320),EAZ09147.1 (Oryza sativa; SEQ ID NO:321), XP_002460236.1 (Sorghumbicolor; SEQ ID NO:322), NP_001146338.1 (Zea mays; SEQ ID NO:323),XP_003519167.1 (Glycine max; SEQ ID NO:324), XP_003550676.1 (Glycinemax; SEQ ID NO:325), XP_003610261.1 (Medicago truncatula; SEQ IDNO:326), XP_003524030.1 (Glycine max; SEQ ID NO:327), XP_003525949.1(Glycine max; SEQ ID NO:328), XP_002325111.1 (Populus trichocarpa; SEQID NO:329), CBI36586.3 (Vitis vinifera; SEQ ID NO:330), XP_002273046.2(Vitis vinifera; SEQ ID NO:331), XP_002303866.1 (Populus trichocarpa;SEQ ID NO:332), and CBI25261.3 (Vitis vinifera; SEQ ID NO:333). Furtherexamples include Sorbi-WRL1 (SEQ ID NO:334), Lupan-WRL1 (SEQ ID NO:335),Ricco-WRL1 (SEQ ID NO:336), and Lupin angustifolius WRI1 (SEQ IDNO:337).

As used herein, the term “monoacylglycerol acyltransferase” or “MGAT”refers to a protein which transfers a fatty acyl group from acyl-CoA toa MAG substrate to produce DAG. Thus, the term “monoacylglycerolacyltransferase activity” at least refers to the transfer of an acylgroup from acyl-CoA to MAG to produce DAG. MGAT is best known for itsrole in fat absorption in the intestine of mammals, where the fattyacids and sn-2 MAG generated from the digestion of dietary fat areresynthesized into TAG in enterocytes for chylomicron synthesis andsecretion. MGAT catalyzes the first step of this process, in which theacyl group from fatty acyl-CoA, formed from fatty acids and CoA, andsn-2 MAG are covalently joined. The term “MGAT” as used herein includesenzymes that act on sn-113 MAG and/or sn-2 MAG substrates to form sn-1,3DAG and/or sn-1,2/2,3-DAG, respectively. In a preferred embodiment, theMGAT has a preference for sn-2 MAG substrate relative to sn-1 MAG, orsubstantially uses only sn-2 MAG as substrate (examples include MGATsdescribed in Cao et al., 2003 (specificity of mouse MGAT1 forsn2-18:1-MAG>sn1/3-18:1-MAG (FIG. 5)); Yen and Farese, 2003 (generalactivities of mouse MGAT1 and human MGAT2 are higher on 2-MAG than on1-MAG acyl-acceptor substrates (FIG. 5); and Cheng et al., 2003(activity of human MGAT3 on 2-MAGs is much higher than on 1/3-MAGsubstrates (FIG. 2D)).

As used herein, MGAT does not include enzymes which transfer an acylgroup preferentially to LysoPA relative to MAG, such enzymes are knownas LPAATs. That is, a MGAT preferentially uses non-phosphorylatedmonoacyl substrates, even though they may have low catalytic activity onLysoPA. A preferred MGAT does not have detectable activity in acylatingLysoPA. As shown herein, a MGAT (i.e., M. musculus MGAT2) may also haveDGAT function but predominantly functions as a MGAT, i.e., it hasgreater catalytic activity as a MGAT than as a DGAT when the enzymeactivity is expressed in units of nmoles product/min/mg protein (alsosee Yen et al., 2002).

There are three known classes of MGAT, referred to as, MGAT1, MGAT2 andMGAT3, respectively. Homologs of the human MGAT1 gene (AF384163; SEQ IDNO:7) are present (i.e. sequences are known) at least in chimpanzee,dog, cow, mouse, rat, zebrafish, Caenorhabditis elegans,Schizosaccharomyces pombe, Saccharomyces cerevisiae, Kluyveromyceslactis, Eremothecium gossypii, Magnaporthe grisea, and Neurosporacrassa. Homologs of the human MGAT2 gene (AY157608) are present at leastin chimpanzee, dog, cow, mouse, rat, chicken, zebrafish, fruit fly, andmosquito. Homologs of the human MGAT3 gene (AY229854) are present atleast in chimpanzee, dog, cow, and zebrafish. However, homologs fromother organisms can be readily identified by methods known in the artfor identifying homologous sequences.

Examples of MGAT1 polypeptides include proteins encoded by MGAT1 genesfrom Homo sapiens (AF384163; SEQ ID NO:7), Mus musculus (AF384162; SEQID NO:8), Pan troglodytes (XM_001166055 and XM_0526044.2; SEQ ID NO:9and SEQ ID NO:10, respectively), Canis familiaris (XM_545667.2; SEQ IDNO:11), Bos taurus (NM_001001153.2; SEQ ID NO:12), Rattus norvegicus(NM_001108803.1; SEQ ID NO:13), Danio rerio MGAT1 (NM_001122623.1; SEQID NO:14), Caenorhabditis elegans (NM_073012.4, NM_182380.5,NM_065258.3, NM_075068.3, and NM_072248.3; SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, respectively),Kluyveromyces lactis (XM_455588.1; SEQ ID NO:20), Ashbya gossypii(NM_208895.1; SEQ ID NO:21), Magnaporthe oryzae (XM_368741.1; SEQ IDNO:22), Ciona intestinalis predicted (XM_002120843.1 SEQ ID NO:23).Examples of MGAT2 polypeptides include proteins encoded by MGAT2 genesfrom Homo sapiens (AY157608; SEQ ID NO:24), Mus musculus (AY157609; SEQID NO:25), Pan troglodytes (XM_522112.2; SEQ ID NO:26), Canis familiaris(XM_542304.1; SEQ ID NO:27), Bos taurus (NM_001099136.1; SEQ ID NO:28),Rattus norvegicus (NM_001109436.2; SEQ ID NO:29), Gallus gallus(XM_424082.2; SEQ ID NO:30), Danio rerio (NM_001006083.1 SEQ ID NO:31),Drosophila melanogaster (NM_136474.2, NM_136473.2, and NM_136475.2; SEQID NO:32, SEQ ID NO:33, and SEQ ID NO:34, respectively), Anophelesgambiae (XM_001688709.1 and XM_315985; SEQ ID NO:35 and SEQ ID NO:36,respectively), Tribolium castaneum (XM_970053.1; SEQ ID NO:37). Examplesof MGAT3 polypeptides include proteins encoded by MGAT3 genes from Homosapiens (AY229854; SEQ ID NO:38), Pan troglodytes (XM_001154107.1,XM_(—) 001154171.1, and XM_527842.2; SEQ ID NO:39, SEQ ID NO:40, and SEQID NO:41), Canis familiaris (XM_845212.1; SEQ ID NO:42), Bos taurus(XM_870406.4; SEQ ID NO:43), Danio rerio (XM_688413.4; SEQ ID NO:44).

As used herein “MGAT pathway” refers to an anabolic pathway, differentto the Kennedy pathway for the formation of TAG, in which DAG is formedby the acylation of either sn-1 MAG or preferably sn-2 MAG, catalysed byMGAT. The DAG may subsequently be used to form TAG or other lipids. TheMGAT pathway is exemplified in FIG. 1.

As used herein, the term “diacylglycerol acyltransferase” (DGAT) refersto a protein which transfers a fatty acyl group from acyl-CoA to a DAGsubstrate to produce TAG. Thus, the term “diacylglycerol acyltransferaseactivity” refers to the transfer of an acyl group from acyl-CoA to DAGto produce TAG. A DGAT may also have MGAT function but predominantlyfunctions as a DGAT, i.e., it has greater catalytic activity as a DGATthan as a MGAT when the enzyme activity is expressed in units of nmolesproduct/min/mg protein (see for example, Yen et al., 2005).

There are three known types of DGAT, referred to as DGAT1, DGAT2 andDGAT3, respectively. DGAT1 polypeptides typically have 10 transmembranedomains, DGAT2 polypeptides typically have 2 transmembrane domains,whilst DGAT3 polypeptides typically have none and are thought to besoluble in the cytoplasm, not integrated into membranes. Examples ofDGAT1 polypeptides include proteins encoded by DGAT1 genes fromAspergillus fumigatus (XP_755172.1; SEQ ID NO:347), Arabidopsis thaliana(CAB44774.1; SEQ ID NO:83), Ricinus communis (AAR11479.1; SEQ IDNO:348), Vernicia fordii (ABC94472.1; SEQ ID NO:349), Vernoniagalamensis (ABV21945.1 and ABV21946.1; SEQ ID NO:350 and SEQ ID NO:351,respectively), Euonymus alatus (AAV31083.1; SEQ ID NO:352),Caenorhabditis elegans (AAF82410.1; SEQ ID NO:353), Rattus norvegicus(NP_445889.1; SEQ ID NO:354), Homo sapiens (NP_036211.2; SEQ ID NO:355),as well as variants and/or mutants thereof. Examples of DGAT2polypeptides include proteins encoded by DGAT2 genes from Arabidopsisthaliana (NP_566952.1; SEQ ID NO:212), Ricinus communis (AAY16324.1; SEQID NO:213), Vernicia fordii (ABC94474.1; SEQ ID NO:214), Mortierellaramanniana (AAK84179.1; SEQ ID NO:215), Homo sapiens (Q96PD7.2; SEQ IDNO:216) (Q58HT5.1; SEQ ID NO:217), Bos taurus (Q70VZ8.1; SEQ ID NO:218),Mus musculus (AAK84175.1; SEQ ID NO:219), as well as variants and/ormutants thereof.

Examples of DGAT3 polypeptides include proteins encoded by DGAT3 genesfrom peanut (Arachis hypogaea, Saha, et al., 2006), as well as variantsand/or mutants thereof. A DGAT has little or no detectable MGATactivity, for example, less than 300 pmol/min/mg protein, preferablyless than 200 pmol/min/mg protein, more preferably less than 100pmol/min/mg protein.

DGAT2 but not DGAT1 shares high sequence homology with the MGAT enzymes,suggesting that DGAT2 and MGAT genes likely share a common geneticorigin. Although multiple isoforms are involved in catalysing the samestep in TAG synthesis, they may play distinct functional roles, assuggested by differential tissue distribution and subcellularlocalization of the DGAT/MGAT family of enzymes. In mammals, MGAT1 ismainly expressed in stomach, kidney, adipose tissue, whilst MGAT2 andMGAT3 show highest expression in the small intestine. In mammals, DGAT1is ubiquitously expressed in many tissues, with highest expression insmall intestine, whilst DGAT2 is most abundant in liver. MGAT3 onlyexists in higher mammals and humans, but not in rodents frombioinformatic analysis. MGAT3 shares higher sequence homology to DGAT2than MGAT1 and MGAT3. MGAT3 exhibits significantly higher DGAT activitythan MGAT1 and MGAT2 enzymes (MGAT3>MGAT1>MGAT2) when either MAGs orDAGs were used as substrates, suggesting MGAT3 functions as a putativeTAG synthase.

Both MGAT1 and MGAT2 belong to the same class of acyltransferases asDGAT2. Some of the motifs that have been shown to be important for DGAT2catalytic activity in some DGAT2s are also conserved in MGATacyltransferases. Of particular interest is a putative neutrallipid-binding domain with the consensus sequence FLXLXXXN (SEQ IDNO:224) where each X is independently any amino acid other than proline,and N is any nonpolar amino acid, located within the N-terminaltransmembrane region followed by a putative glycerol/phospholipidacyltransferase domain. The FLXLXXXN motif (SEQ ID NO:224) is found inthe mouse DGAT2 (amino acids 81-88) and MGAT1/2 but not in yeast orplant DGAT2s. It is important for activity of the mouse DGAT2. OtherDGAT2 and/or MGAT1/2 sequence motifs include:

1. A highly conserved YFP tripeptide (SEQ ID NO:220) in most DGAT2polypeptides and also in MGAT1 and MGAT2, for example, present as aminoacids 139-141 in mouse DGAT2. Mutating this motif within the yeast DGAT2with non-conservative substitutions rendered the enzyme non-functional.2. HPHG tetrapeptide (SEQ ID NO:221), highly conserved in MGATs as wellas in DGAT2 sequences from animals and fungi, for example, present asamino acids 161-164 in mouse DGAT2, and important for catalytic activityat least in yeast and mouse DGAT2. Plant DGAT2 acyltransferases have aEPHS (SEQ ID NO:222) conserved sequence instead, so conservative changesto the first and fourth amino acids can be tolerated.3. A longer conserved motif which is part of the putative glycerolphospholipid domain. An example of this motif isRXGFX(K/R)XAXXXGXXX(L/V)VPXXXFG(E/Q) (SEQ ID NO:223), which is presentas amino acids 304-327 in mouse DGAT2. This motif is less conserved inamino acid sequence than the others, as would be expected from itslength, but homologs can be recognised by motif searching. The spacingmay vary between the more conserved amino acids, i.e., there may beadditional X amino acids within the motif, or less X amino acidscompared to the sequence above.

As used herein, the term “glycerol-3-phosphate acyltransferase” or“GPAT” refers to a protein which acylates glycerol-3-phosphate (G-3-P)to form LysoPA and/or MAG, the latter product forming if the GPAT alsohas phosphatase activity on LysoPA. The acyl group that is transferredis typically from acyl-CoA. Thus, the term “glycerol-3-phosphateacyltransferase activity” refers to the acylation of G-3-P to formLysoPA and/or MAG. The term “GPAT” encompasses enzymes that acylateG-3-P to form sn-1 LPA and/or sn-2 LPA, preferably sn-2 LPA. In apreferred embodiment, the GPAT has phosphatase activity. In a mostpreferred embodiment, the GPAT is a sn-2 GPAT having phosphataseactivity which produces sn-2 MAG.

As used herein, the term “sn-1 glycerol-3-phosphate acyltransferase”(sn-1 GPAT) refers to a protein which acylates sn-glycerol-3-phosphate(G-3-P) to preferentially form 1-acyl-sn-glycerol-3-phosphate (sn-1LPA). Thus, the term “sn-1 glycerol-3-phosphate acyltransferaseactivity” refers to the acylation of sn-glycerol-3-phosphate to form1-acyl-sn-glycerol-3-phosphate (sn-1 LPA).

As used herein, the term “sn-2 glycerol-3-phosphate acyltransferase”(sn-2 GPAT) refers to a protein which acylates sn-glycerol-3-phosphate(G-3-P) to preferentially form 2-acyl-sn-glycerol-3-phosphate (sn-2LPA). Thus, the term “sn-2 glycerol-3-phosphate acyltransferaseactivity” refers to the acylation of sn-glycerol-3-phosphate to form2-acyl-sn-glycerol-3-phosphate (sn-2 LPA).

The GPAT family is large and all known members contain two conserveddomains, a plsC acyltransferase domain (PF01553; SEQ ID NO:225) and aHAD-like hydrolase (PF12710; SEQ ID NO:226) superfamily domain. Inaddition to this, in Arabidopsis thaliana, GPAT4-8 all contain aN-terminal region homologous to a phosphoserine phosphatase domain(PF00702; SEQ ID NO:227). GPAT4 and GPAT6 both contain conservedresidues that are known to be critical to phosphatase activity,specifically conserved amino acids (shown in bold) in Motif I(DXDX[T/V][L/V]; SEQ ID NO:229) and Motif III (K-[G/S][D/S]XXX[D/N]; SEQID NO:330) located at the N-terminus (Yang et al., 2010). Preferably,the GPAT has sn-2 preference and phosphatase activity to produce sn-2MAG (also referred to herein as “2-MAG”) from glycerol-3-phosphate(G-3-P) (FIG. 1), for example, GPAT4 (NP_171667.1; SEQ ID NO:144) andGPAT6 (NP_181346.1; SEQ ID NO:145) from Arabidopsis. More preferably,the GPAT uses acyl-CoA as a fatty acid substrate.

Homologues of GPAT4 (NP_171667.1; SEQ ID NO:144) and GPAT6 (NP_181346.1;SEQ ID NO:145) include AAF02784.1 (Arabidopsis thaliana; SEQ ID NO:146),AAL32544.1 (Arabidopsis thaliana; SEQ ID NO:147), AAP03413.1 (Oryzasativa; SEQ ID NO:148), ABK25381.1 (Picea sitchensis; SEQ ID NO:149),ACN34546.1 (Zea Mays; SEQ ID NO:150), BAF00762.1 (Arabidopsis thaliana;SEQ ID NO:151), BAH00933.1 (Oryza sativa; SEQ ID NO:152), EAY84189.1(Oryza sativa; SEQ ID NO:153), EAY98245.1 (Oryza sativa; SEQ ID NO:154),EAZ21484.1 (Oryza sativa; SEQ ID NO:155), EEC71826.1 (Oryza sativa; SEQID NO:156), EEC76137.1 (Oryza sativa; SEQ ID NO:157), EEE59882.1 (Oryzasativa; SEQ ID NO:158), EFJ08963.1 (Selaginella moellendorffii; SEQ IDNO:159), EFJ08964.1 (Selaginella moellendorffii; SEQ ID NO:160),EFJ11200.1 (Selaginella moellendorffii; SEQ ID NO:161), EFJ15664.1(Selaginella moellendorffii; SEQ ID NO:162), EFJ24086.1 (Selaginellamoellendorffii; SEQ ID NO:163), EFJ29816.1 (Selaginella moellendorffii;SEQ ID NO:164), EFJ29817.1 (Selaginella moellendorifii; SEQ ID NO:165),NP_001044839.1 (Oryza sativa; SEQ ID NO:166), NP_001045668.1 (Oryzasativa; SEQ ID NO:167), NP_001147442.1 (Zea mays; SEQ ID NO:168),NP_001149307.1 (Zea mays; SEQ ID NO:169), NP_001168351.1 (Zea mays; SEQID NO:170), AFH02724.1 (Brassica napus; SEQ ID NO:171) NP_191950.2(Arabidopsis thaliana; SEQ ID NO:172), XP_001765001.1 (Physcomitrellapatens; SEQ ID NO:173), XP_001769671.1 (Physcomitrella patens; SEQ IDNO:174), XP_001769724.1 (Physcomitrella patens; SEQ ID NO:175),XP_001771186.1 (Physcomitrella patens; SEQ ID NO:176), XP_001780533.1(Physcomitrella patens; SEQ ID NO:177), XP_002268513.1 (Vitis vinifera;SEQ ID NO:178), XP_002275348.1 (Vitis vinifera; SEQ ID NO:179),XP_002276032.1 (Vitis vinifera; SEQ ID NO:180), XP_002279091.1 (Vitisvinifera; SEQ ID NO:181), XP_002309124.1 (Populus trichocarpa; SEQ IDNO:182), XP_002309276.1 (Populus trichocarpa; SEQ ID NO:183),XP_002322752.1 (Populus trichocarpa; SEQ ID NO:184), XP_002323563.1(Populus trichocarpa; SEQ ID NO:185), XP_002439887.1 (Sorghum bicolor;SEQ ID NO:186), XP_002458786.1 (Sorghum bicolor; SEQ ID NO:187),XP_002463916.1 (Sorghum bicolor; SEQ ID NO:188), XP_002464630.1 (Sorghumbicolor; SEQ ID NO:189), XP_002511873.1 (Ricinus communis; SEQ IDNO:190), XP_002517438.1 (Ricinus communis; SEQ ID NO:191),XP_002520171.1 (Ricinus communis; SEQ ID NO:192), XP_002872955.1(Arabidopsis lyrata; SEQ ID NO:193), XP_002881564.1 (Arabidopsis lyrata;SEQ ID NO:194), ACT32032.1 (Vernicia fordii; SEQ ID NO:195),NP_001051189.1 (Oryza sativa; SEQ ID NO:196), AFH02725.1 (Brassicanapus; SEQ ID NO:197), XP_002320138.1 (Populus trichocarpa; SEQ IDNO:198), XP_002451377.1 (Sorghum bicolor; SEQ ID NO:199), XP_002531350.1(Ricinus communis; SEQ ID NO:200), and XP_002889361.1 (Arabidopsislyrata; SEQ ID NO:201).

Conserved motifs and/or residues can be used as a sequence-baseddiagnostic for the identification of bifunctional GPAT/phosphataseenzymes. Alternatively, a more stringent function-based assay could beutilised. Such an assay involves, for example, feeding labelledglycerol-3-phosphate to cells or microsomes and quantifying the levelsof labelled products by thin-layer chromatography or a similartechnique. GPAT activity results in the production of labelled LPAwhilst GPAT/phosphatase activity results in the production of labelledMAG.

As used herein, the term “Oleosin” refers to an amphipathic proteinpresent in the membrane of oil bodies in the storage tissues of seeds(see, for example, Huang, 1996; Lin et al., 2005; Capuano et al., 2007;Lui et al., 2009; Shimada and Hara-Nishimura, 2010).

Plant seeds accumulate TAG in subcellular structures called oil bodies.These organelles consist of a TAG core surround by a phospholipidmonolayer containing several embedded proteins including oleosins(Jolivet et al., 2004). Oleosins represent the most abundant protein inthe membrane of oil bodies.

Oleosins are of low M_(r) (15-26,000). Within each seed species, thereare usually two or more oleosins of different M_(r). Each oleosinmolecule contains a relatively hydrophilic N-terminal domain (forexample, about 48 amino acid residues), a central totally hydrophobicdomain (for example, of about 70-80 amino acid residues) which isparticularly rich in aliphatic amino acids such as alanine, glycine,leucine, isoleucine and valine, and an amphipathic α-helical domain (forexample, about of about 33 amino acid residues) at or near theC-terminus. Generally, the central stretch of hydrophobic residues isinserted into the lipid core and the amphiphatic N-terminal and/oramphiphatic C-terminal are located at the surface of the oil bodies,with positively charged residues embedded in a phospholipid monolayerand the negatively charged ones exposed to the exterior.

As used herein, the term “Oleosin” encompasses polyoleosins which havemultiple oleosin polypeptides fused together as a single polypeptide,for example 2×, 4× or 6× oleosin peptides, and caleosins which bindcalcium (Froissard et al., 2009), and steroleosins which bind sterols.However, generally a large proportion of the oleosins of oil bodies willnot be caleosins and/or steroleosins.

A substantial number of oleosin protein sequences, and nucleotidesequences encoding therefor, are known from a large number of differentplant species. Examples include, but are not limited to, oleosins fromArabidposis, canola, corn, rice, peanut, castor, soybean, flax, grape,cabbage, cotton, sunflower, sorghum and barley. Examples of oleosins(with their Accession Nos) include Brassica napus oleosin (CAA57545.1;SEQ ID NO:362), Brassica napus oleosin S1-1 (ACG69504.1; SEQ ID NO:363),Brassica napus oleosin S2-1 (ACG69503.1; SEQ ID NO:364), Brassica napusoleosin S3-1 (ACG69513.1; SEQ ID NO:365), Brassica napus oleosin S4-1(ACG69507.1; SEQ ID NO:366), Brassica napus oleosin S5-1 (ACG69511.1;SEQ ID NO:367), Arachis hypogaea oleosin 1 (AAZ20276.1; SEQ ID NO:368),Arachis hypogaea oleosin 2 (AAU21500.1; SEQ ID NO:369), Arachis hypogaeaoleosin 3 (AAU21501.1; SEQ ID NO:370), Arachis hypogaea oleosin 5(ABC96763.1; SEQ ID NO:371), Ricinus communis oleosin 1 (EEF40948.1; SEQID NO:372), Ricinus communis oleosin 2 (EEF51616.1; SEQ ID NO:373),Glycine max oleosin isoform a (P29530.2; SEQ ID NO:374), Glycine maxoleosin isoform b (P29531.1; SEQ ID NO:375), Linum usitatissimum oleosinlow molecular weight isoform (ABB01622.1; SEQ ID NO:376), Linumusitatissimum oleosin high molecular weight isoform (ABB01624.1; SEQ IDNO:377), Helianthus annuus oleosin (CAA44224.1; SEQ ID NO:378), Zea maysoleosin (NP_001105338.1; SEQ ID NO:379), Brassica napus steroleosin(ABM30178.1; SEQ ID NO:380), Brassica napus steroleosin SLO1-1(ACG69522.1; SEQ ID NO:381), Brassica napus steroleosin SLO2-1(ACG69525.1; SEQ ID NO:382), Sesamum indicum steroleosin (AAL13315.1;SEQ ID NO:383), Zea mays steroleosin (NP_001152614.1; SEQ ID NO:384),Brassica napus caleosin CLO-1 (ACG69529.1; SEQ ID NO:385), Brassicanapus caleosin CLO-3 (ACG69527.1; SEQ ID NO:386), Sesamum indicumcaleosin (AAF13743.1; SEQ ID NO:387), Zea mays caleosin (NP_001151906.1;SEQ ID NO:388).

As used herein, the term a “polypeptide involved in starch biosynthesis”refers to any polypeptide, the downregulation of which in a cell belownormal (wild-type) levels results in a reduction in the level of starchsynthesis and an increase in the levels of starch. An example of such apolypeptide is AGPase.

As used herein, the term “ADP-glucose phosphorylase” or “AGPase” refersto an enzyme which regulates starch biosynthesis, catalysing conversionof glucose-1-phosphate and ATP to ADP-glucose which serves as thebuilding block for starch polymers. The active form of the AGPase enzymeconsists of 2 large and 2 small subunits.

The ADPase enzyme in plants exists primarily as a tetramer whichconsists of 2 large and 2 small subunits. Although these subunits differin their catalytic and regulatory roles depending on the species (Kuhnet al., 2009), in plants the small subunit generally displays catalyticactivity. The molecular weight of the small subunit is approximately50-55 kDa. The molecular weight of the large subunit is approximately55-60 kDa. The plant enzyme is strongly activated by 3-phosphoglycerate(PGA), a product of carbon dioxide fixation; in the absence of PGA, theenzyme exhibits only about 3% of its activity. Plant AGPase is alsostrongly inhibited by inorganic phosphate (Pi). In contrast, bacterialand algal AGPase exist as homotetramers of 50 kDa. The algal enzyme,like its plant counterpart, is activated by PGA and inhibited by Pi,whereas the bacterial enzyme is activated by fructose-1,6-bisphosphate(FBP) and inhibited by AMP and Pi.

As used herein, the term “polypeptide involved in the degradation oflipid and/or which reduces lipid content” refers to any polypeptide, thedownregulation of which in a cell below normal (wild-type) levelsresults an increase in the level of oil, such as fatty acids and/orTAGs, in the cell, preferably a cell of vegetative tissue of a plant.Examples of such polypeptides include, but are not limited, lipases, ora lipase such as CGi58 polypeptide, SUGAR-DEPENDENT1 triacylglycerollipase (see, for example, Kelly et al., 2012) or a lipase described inWO 2009/027335.

As used herein, the term “lipase” refers to a protein which hydrolyzesfats into glycerol and fatty acids. Thus, the term “lipase activity”refers to the hydrolysis of fats into glycerol and fatty acids.

As used herein, the term “CGi58” refers to a soluble acyl-CoA-dependentlysophosphatidic acid acyltransferase also known in the art as“At4g24160” (in plants) and “Ict1p” (in yeast). The plant gene such asthat from Arabidopsis gene locus, At4g24160, is expressed as twoalternative transcripts: a longer full-length isoform (At4g24160.1) anda smaller isoform (At4g24160.2) missing a portion of the 3′ end (seeJames et al., 2010; Ghosh et al., 2009; US 201000221400). Both mRNAscode for a protein that is homologous to the human CGI-58 protein andother orthologous members of this α/β hydrolase family (ABHD). In anembodiment, the CGI58 (At4g24160) protein contains three motifs that areconserved across plant species: a GXSXG lipase motif (SEQ ID NO:419), aHX(4)D acyltransferase motif (SEQ ID NO:420), and VX(3)HGF, a probablelipid binding motif (SEQ ID NO:421). The human CGI-58 protein haslysophosphatidic acid acyltransferase (LPAAT) activity but not lipaseactivity. In contrast, the plant and yeast proteins possess a canonicallipase sequence motif GXSXG (SEQ ID NO:419), that is absent fromvertebrate (humans, mice, and zebrafish) proteins. Although the plantand yeast CGI58 proteins appear to possess detectable amounts of TAGlipase and phospholipase A activities in addition to LPAAT activity, thehuman protein does not.

Disruption of the homologous CGI-58 gene in Arabidopsis thaliana resultsin the accumulation of neutral lipid droplets in mature leaves. Massspectroscopy of isolated lipid droplets from cgi-58 loss-of-functionmutants showed they contain triacylglycerols with common leaf-specificfatty acids. Leaves of mature cgi-58 plants exhibit a marked increase inabsolute triacylglycerol levels, more than 10-fold higher than inwild-type plants. Lipid levels in the oil-storing seeds of cgi-58loss-of-function plants were unchanged, and unlike mutations inβ-oxidation, the cgi-58 seeds germinated and grew normally, requiring norescue with sucrose (James et al., 2010).

Examples of CGi58 polypeptides include proteins from Arabidopsisthaliana (NP_194147.2; SEQ ID NO:429), Brachypodium distachyon(XP_003578450.1; SEQ ID NO:430), Glycine max (XP_003523638.1; SEQ IDNO:431), Zea mays (NP_001149013.1; SEQ ID NO:432), Sorghum bicolor(XP_002460538.1; SEQ ID NO:433), Ricinus communis (XP_002510485.1; SEQID NO:434), Medicago truncatula (XP_003603733.1; SEQ ID NO:435), andOryza sativa (EAZ09782.1; SEQ ID NO:436).

As used herein, the term “Leafy Cotyledon 2” or “LEC2” refers to a B3domain transcription factor which participates in zygotic and in somaticembryogenesis. Its ectopic expression facilitates the embryogenesis fromvegetative plant tissues (Alemanno et al., 2008). LEC2 also comprises aDNA binding region found thus far only in plant proteins. Examples ofLEC2 polypeptides include proteins from Arabidopsis thaliana(NP_564304.1) (SEQ ID NO:442), Medicago truncatula (CAA42938.1) (SEQ IDNO:443) and Brassica napus (ADO16343.1) (SEQ ID NO:444).

As used herein, the term “BABY BOOM” or “BBM” refers an AP2/ERFtranscription factor that induces regeneration under culture conditionsthat normally do not support regeneration in wild-type plants. Ectopicexpression of Brassica napus BBM (BnBBM) genes in B. napus andArabidopsis induces spontaneous somatic embryogenesis and organogenesisfrom seedlings grown on hormone-free basal medium (Boutilier et al.,2002). In tobacco, ectopic BBM expression is sufficient to induceadventitious shoot and root regeneration on basal medium, but exogenouscytokinin is required for somatic embryo (SE) formation (Srinivasan etal., 2007). Examples of BBM polypeptides include proteins fromArabidopsis thaliana (NP_197245.2) (SEQ ID NO:445) and Medicagotruncatula (AAW82334.1) (SEQ ID NO:446).

As used herein, the term “FAD2” refers to a membrane bound delta-12fatty acid desturase that desaturates oleic acid (18:1^(Δ9)) to producelinoleic acid (C18:2^(Δ9,12)).

As used herein, the term “epoxygenase” or “fatty acid epoxygenase”refers to an enzyme that introduces an epoxy group into a fatty acidresulting in the production of an epoxy fatty acid. In preferredembodiment, the epoxy group is introduced at the 12th carbon on a fattyacid chain, in which case the epoxygenase is a Δ12-epoxygenase,especially of a C16 or C18 fatty acid chain. The epoxygenase may be aΔ9-epoxygenase, a Δ15 epoxygenase, or act at a different position in theacyl chain as known in the art. The epoxygenase may be of the P450class. Preferred epoxygenases are of the mono-oxygenase class asdescribed in WO98/46762. Numerous epoxygenases or presumed epoxygenaseshave been cloned and are known in the art. Further examples ofexpoxygenases include proteins comprising an amino acid sequenceprovided in SEQ ID NO:21 of WO 2009/129582, polypeptides encoded bygenes from Crepis paleastina (CAA76156, Lee et al., 1998), Stokesialaevis (AAR23815, Hatanaka et al., 2004) (monooxygenase type), Euphorbialagascae (AAL62063) (P450 type), human CYP2J2 (arachidonic acidepoxygenase, U37143); human CYPIA1 (arachidonic acid epoxygenase,K03191), as well as variants and/or mutants thereof.

As used herein, the term, “hydroxylase” or “fatty acid hydroxylase”refers to an enzyme that introduces a hydroxyl group into a fatty acidresulting in the production of a hydroxylated fatty acid. In a preferredembodiment, the hydroxyl group is introduced at the 2nd, 12th and/or17th carbon on a C18 fatty acid chain. Preferably, the hydroxyl group isintroduced at the 12^(th) carbon, in which case the hydroxylase is aΔ12-hydroxylase. In another preferred embodiment, the hydroxyl group isintroduced at the 15th carbon on a C16 fatty acid chain. Hydroxylasesmay also have enzyme activity as a fatty acid desaturase. Examples ofgenes encoding Δ12-hydroxylases include those from Ricinus communis(AAC9010, van de Loo 1995); Physaria lindheimeri, (ABQ01458, Dauk etal., 2007); Lesquerella fendleri, (AAC32755, Broun et al., 1998); Daucuscarota, (AAK30206); fatty acid hydroxylases which hydroxylate theterminus of fatty acids, for example: A, thaliana CYP86A1 (P48422, fattyacid ω-hydroxylase); Vicia sativa CYP94A1 (P98188, fatty acidω-hydroxylase); mouse CYP2E1 (X62595, lauric acid ω-1 hydroxylase); ratCYP4A1 (M57718, fatty acid ω-hydroxylase), as well as variants and/ormutants thereof.

As used herein, the term “conjugase” or “fatty acid conjugase” refers toan enzyme capable of forming a conjugated bond in the acyl chain of afatty acid. Examples of conjugases include those encoded by genes fromCalendula officinalis (AF343064, Qiu et al., 2001); Vernicia fordii(AAN87574, Dyer et al., 2002); Punica granatum (AY178446, Iwabuchi etal., 2003) and Trichosanthes kirilowii (AY178444, Iwabuchi et al.,2003); as well as variants and/or mutants thereof.

As used herein, the term “acetylenase” or “fatty acid acetylenase”refers to an enzyme that introduces a triple bond into a fatty acidresulting in the production of an acetylenic fatty acid. In a preferredembodiment, the triple bond is introduced at the 2nd, 6th, 12th and/or17th carbon on a C18 fatty acid chain. Examples acetylenases includethose from Helianthus annuus (AA038032, ABC59684), as well as variantsand/or mutants thereof.

Examples of such fatty acid modifying genes include proteins accordingto the following Accession Numbers which are grouped by putativefunction, and homologues from other species: Δ12 acetylenases ABC00769,CAA76158, AAO38036, AAO38032; Δ12 conjugases AAG42259, AAG42260,AAN87574; Δ12 desaturases P46313, ABS18716, AAS57577, AAL61825,AAF04093, AAF04094; Δ12 epoxygenases XP_001840127, CAA76156, AAR23815;Δ12 hydroxylases ACF37070, AAC32755, ABQ01458, AAC49010; and Δ12 P450enzymes such as AF406732.

As used herein, the term “vegetative tissue” or “vegetative plant part”is any plant tissue, organ or part other than organs for sexualreproduction of plants, specifically seed bearing organs, flowers,pollen, fruits and seeds. Vegetative tissues and parts include at leastplant leaves, stems (including bolts and tillers but excluding theheads), tubers and roots, but excludes flowers, pollen, seed includingthe seed coat, embryo and endosperm, fruit including mesocarp tissue,seed-bearing pods and seed-bearing heads. In one embodiment, thevegetative part of the plant is an aerial plant part. In another orfurther embodiment, the vegetative plant part is a green part such as aleaf or stem.

As used herein, the term “wild-type” or variations thereof refers to acell, or non-human organism or part thereof that has not beengenetically modified.

The term “corresponding” refers to a vegetative plant part, a cell, ornon-human organism or part thereof, or seed that has the same or similargenetic background as a vegetative plant part, a cell, or non-humanorganism or part thereof, or seed of the invention but that has not beenmodified as described herein (for example, a vegetative plant part, acell, or non-human organism or part thereof, or seed lacks an exogenouspolynucleotide encoding a MGAT or an exogenous MGAT). In a preferredembodiment, a vegetative plant part, a cell, or non-human organism orpart thereof, or seed is at the same developmental stage as a vegetativeplant part, a cell, or non-human organism or part thereof, or seed ofthe invention. For example, if the non-human organism is a floweringplant, then preferably the corresponding plant is also flowering. Acorresponding a vegetative plant part, a cell, or non-human organism orpart thereof, or seed can be used as a control to compare levels ofnucleic acid or protein expression, or the extent and nature of traitmodification, for example non-polar lipid production and/or content,with a vegetative plant part, a cell, or non-human organism or partthereof, or seed modified as described herein. A person skilled in theart is readily able to determine an appropriate “corresponding” cell,tissue, organ or organism for such a comparison.

As used herein “compared with” refers to comparing levels of a non-polarlipid or total non-polar lipid content of the transgenic non-humanorganism or part thereof expressing the one or more exogenouspolynucleotides or exogenous polypeptides with a transgenic non-humanorganism or part thereof lacking the one or more exogenouspolynucleotides or polypeptides.

As used herein, “enhanced ability to produce non-polar lipid” is arelative term which refers to the total amount of non-polar lipid beingproduced by a cell, or non-human organism or part thereof of theinvention being increased relative to a corresponding cell, or non-humanorganism or part thereof. In one embodiment, the TAG and/orpolyunsaturated fatty acid content of the non-polar lipid is increased.

As used herein, “germinate at a rate substantially the same as for acorresponding wild-type plant” refers to seed of a plant of theinvention being relatively fertile when compared to seed of a wild typeplant lacking the defined exogenous polynucleotide(s). In oneembodiment, the number of seeds which germinate, for instance when grownunder optimal greenhouse conditions for the plant species, is at least75%, more preferably at least 90%, of that when compared tocorresponding wild-type seed. In another embodiment, the seeds whichgerminate, for instance when grown under optimal greenhouse conditionsfor the plant species, grow at a rate which, on average, is at least75%, more preferably at least 90%, of that when compared tocorresponding wild-type plants.

As used herein, the term “an isolated or recombinant polynucleotidewhich down regulates the production and/or activity of an endogenousenzyme” or variations thereof, refers to a polynucleotide that encodesan RNA molecule that down regulates the production and/or activity (forexample, encoding an siRNA, hpRNAi), or itself down regulates theproduction and/or activity (for example, is an siRNA which can bedelivered directly to, for example, a cell) of an endogenous enzyme forexample, DGAT, sn-1 glycerol-3-phosphate acyltransferase (GPAT),1-acyl-glycerol-3-phosphate acyltransferase (LPAAT),acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT), phosphatidicacid phosphatase (PAP), AGPase, or delta-12 fatty acid desturase (FAD2),or a combination of two or more thereof.

As used herein, the term “on a weight basis” refers to the weight of asubstance (for example, TAG, DAG, fatty acid) as a percentage of theweight of the composition comprising the substance (for example, seed,leaf). For example, if a transgenic seed has 25 μg total fatty acid per120 μg seed weight; the percentage of total fatty acid on a weight basisis 20.8%.

As used herein, the term “on a relative basis” refers to the amount of asubstance in a composition comprising the substance in comparison with acorresponding composition, as a percentage.

As used herein, the term “the relative non-lipid content” refers to theexpression of the non-polar lipid content of a cell, organism or partthereof, or extracted lipid therefrom, in comparison with acorresponding cell, organism or part thereof, or the lipid extractedfrom the corresponding cell, organism or part thereof, as a percentage.For example, if a transgenic seed has 25 μg total fatty acid, whilst thecorresponding seed had 20 μg total fatty acid; the increase in non-polarlipid content on a relative basis equals 25%.

As used herein, the term “biofuel” refers to any type of fuel, typicallyas used to power machinery such as automobiles, trucks or petroleumpowered motors, whose energy is derived from biological carbon fixation.Biofuels include fuels derived from biomass conversion, as well as solidbiomass, liquid fuels and biogases. Examples of biofuels includebioalcohols, biodiesel, synthetic diesel, vegetable oil, bioethers,biogas, syngas, solid biofuels, algae-derived fuel, biohydrogen,biomethanol, 2,5-Dimethylfuran (DMF), biodimethyl ether (bioDME),Fischer-Tropsch diesel, biohydrogen diesel, mixed alcohols and wooddiesel.

As used herein, the term “bioalcohol” refers to biologically producedalcohols, for example, ethanol, propanol and butanol. Bioalcohols areproduced by the action of microorganisms and/or enzymes through thefermentation of sugars, hemicellulose or cellulose.

As used herein, the term “biodiesel” refers to a composition comprisingfatty acid methyl- or ethyl-esters derived from non-polar lipids bytransesterification.

As used herein, the term “synthetic diesel” refers to a form of dieselfuel which is derived from renewable feedstock rather than the fossilfeedstock used in most diesel fuels.

As used herein, the term “vegetable oil” includes a pure plant oil (orstraight vegetable oil) or a waste vegetable oil (by product of otherindustries).

As used herein, the term ‘bioethers” refers to compounds that act asoctane rating enhancers.

As used herein, the term “biogas” refers to methane or a flammablemixture of methane and other gases produced by anaerobic digestion oforganic material by anaerobes.

As used herein, the term “syngas” refers to a gas mixture that containsvarying amounts of carbon monoxide and hydrogen and possibly otherhydrocarbons, produced by partial combustion of biomass.

As used herein, the term “solid biofuels” includes wood, sawdust, grasstrimming, and non-food energy crops.

As used herein, the term “cellulosic ethanol” refers to ethanol producedfrom cellulose or hemicellulose.

As used herein, the term “algae fuel” refers to a biofuel made fromalgae and includes algal biodiesel, biobutanol, biogasoline, methane,ethanol, and the equivalent of vegetable oil made from algae.

As used herein, the term “biohydrogen” refers to hydrogen producedbiologically by, for example, algae.

As used herein, the term “biomethanol’ refers to methanol producedbiologically. Biomethanol may be produced by gasification of organicmaterials to syngas followed by conventional methanol synthesis.

As used herein, the term “2,5-Dimethylfuran” or “DMF” refers to aheterocyclic compound with the formula (CH₃)₂C₄H₂O. DMF is a derivativeof furan that is derivable from cellulose.

As used herein, the term “biodimethyl ether” or “bioDME”, also known asmethoxymethane, refers to am organic compound with the formula CH₃OCH₃.Syngas may be converted into methanol in the presence of catalyst(usually copper-based), with subsequent methanol dehydration in thepresence of a different catalyst (for example, silica-alumina) resultingin the production of DME.

As used herein, the term “Fischer-Tropsch” refers to a set of chemicalreactions that convert a mixture of carbon monoxide and hydrogen intoliquid hydrocarbons. The syngas can first be conditioned using forexample, a water gas shift to achieve the required H₂/CO ratio. Theconversion takes place in the presence of a catalyst, usually iron orcobalt. The temperature, pressure and catalyst determine whether a lightor heavy syncrude is produced. For example at 330° C. mostly gasolineand olefins are produced whereas at 180° to 250° C. mostly diesel andwaxes are produced. The liquids produced from the syngas, which comprisevarious hydrocarbon fractions, are very clean (sulphur free)straight-chain hydrocarbons. Fischer-Tropsch diesel can be produceddirectly, but a higher yield is achieved if first Fischer-Tropsch wax isproduced, followed by hydrocracking.

As used herein, the term “biochar” refers to charcoal made from biomass,for example, by pyrolysis of the biomass.

As used herein, the term “feedstock” refers to a material, for example,biomass or a conversion product thereof (for example, syngas) when usedto produce a product, for example, a biofuel such as biodiesel or asynthetic diesel.

As used herein, the term “industrial product” refers to a hydrocarbonproduct which is predominantly made of carbon and hydrogen such as fattyacid methyl- and/or ethyl-esters or alkanes such as methane, mixtures oflonger chain alkanes which are typically liquids at ambienttemperatures, a biofuel, carbon monoxide and/or hydrogen, or abioalcohol such as ethanol, propanol, or butanol, or biochar. The term“industrial product” is intended to include intermediary products thatcan be converted to other industrial products, for example, syngas isitself considered to be an industrial product which can be used tosynthesize a hydrocarbon product which is also considered to be anindustrial product. The term industrial product as used herein includesboth pure forms of the above compounds, or more commonly a mixture ofvarious compounds and components, for example the hydrocarbon productmay contain a range of carbon chain lengths, as well understood in theart.

As used herein, “gloss” refers to an optical phenomenon caused whenevaluating the appearance of a surface. The evaluation of glossdescribes the capacity of a surface to reflect directed light.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

The term “and/or”, e.g., “X and/or Y” shall be understood to mean either“X and Y” or “X or Y” and shall be taken to provide explicit support forboth meanings or for either meaning.

As used herein, the term about, unless stated to the contrary, refers to+/−10%, more preferably +/−5%, more preferably +/−2%, more preferably+/−1%, even more preferably +/−0.5%, of the designated value.

Production of Diacylgylerols and Triacylglycerols

In one embodiment, the vegetative plant part, transgenic non-humanorganism or part thereof of the invention produces higher levels ofnon-polar lipids such as DAG or TAG, preferably both, than acorresponding vegetative plant part, non-human organism or part thereof.In one example, transgenic plants of the invention produce seeds,leaves, leaf portions of at least 1 cm² in surface area, stems and/ortubers having an increased non-polar lipid content such as DAG or TAG,preferably both, when compared to corresponding seeds, leaves, leafportions of at least 1 cm² in surface area, stems or tubers. Thenon-polar lipid content of the vegetative plant part, non-human organismor part thereof is at 0.5% greater on a weight basis when compared to acorresponding non-human organism or part thereof, or as further definedin Feature (i).

In another embodiment, the vegetative plant part, transgenic non-humanorganism or part thereof, preferably a plant or seed, produce DAGsand/or TAGs that are enriched for one or more particular fatty acids. Awide spectrum of fatty acids can be incorporated into DAGs and/or TAGs,including saturated and unsaturated fatty acids and short-chain andlong-chain fatty acids. Some non-limiting examples of fatty acids thatcan be incorporated into DAGs and/or TAGs and which may be increased inlevel include: capric (10:0), lauric (12:0), myristic (14:0), palmitic(16:0), palmitoleic (16:1), stearic (18:0), oleic (18:1), vaccenic(18:1), linoleic (18:2), eleostearic (18:3), γ-linolenic (18:3),α-linolenic (18:3ω3), stearidonic (18:4ω3), arachidic (20:0),eicosadienoic (20:2), dihomo-γ-linoleic (20:3), eicosatrienoic (20:3),arachidonic (20:4), eicosatetraenoic (20:4), eicosapentaenoic (20:5ω3),behenic (22:0), docosapentaenoic (22:5ω), docosahexaenoic (22:6ω3),lignoceric (24:0), nervonic (24:1), cerotic (26:0), and montanic (28:0)fatty acids. In one embodiment of the present invention, the vegetativeplant part, transgenic organism or parts thereof is enriched for DAGsand/or TAGs comprising oleic acid, or polyunsaturated fatty acids.

In one embodiment of the invention, the vegetative plant part,transgenic non-human organism or part thereof, preferably a plant orseed, is transformed with a chimeric DNA which encodes an MGAT which mayor may not have DGAT activity. Expression of the MGAT preferably resultsin higher levels of non-polar lipids such as DAG or TAG and/or increasednon-polar lipid yield in said vegetative plant part, transgenicnon-human organism or part thereof. In a preferred embodiment, thetransgenic non-human organism is a plant.

In a further embodiment, the vegetative plant part, transgenic non-humanorganism or part thereof is transformed with a chimeric DNA whichencodes a GPAT or a DGAT. Preferably, the vegetative plant part ortransgenic non-human organism is transformed with both chimeric DNAs,which are preferably covalently linked on one DNA molecule such as, forexample, a single T-DNA molecule.

Yang et al. (2010) describe two glycerol-3-phosphate acyltransferases(GPAT4 and GPAT6) from Arabidopsis with sn-2 preference and phosphataseactivity that are able to produce sn-2 MAG from glycerol-3-phosphate(G-3-P) (FIG. 1). These enzymes are proposed to be part of the cutinsynthesis pathway. Arabidopsis GPAT4 and GPAT6 have been shown to useacyl-CoA as a fatty acid substrate (Zheng et al., 2003).

Combining a bifunctional GPAT/phosphatase with a MGAT yields a novel DAGsynthesis pathway using G-3-P as one substrate and two acyl groupsderived from acyl-CoA as the other substrates. Similarly, combining sucha bifunctional GPAT/phosphatase with a MGAT which has DGAT activityyields a novel TAG synthesis pathway using glycerol-3-phosphate as onesubstrate and three acyl groups derived from acyl-CoA as othersubstrates.

Accordingly, in one embodiment of the invention, the vegetative plantpart, transgenic non-human organism or part thereof is co-transformedwith a bifunctional GPAT/phosphatase and with a MGAT which does not haveDGAT activity. This would result in the production of MAG by thebifunctional GPAT/phosphatase which would then be converted to DAG bythe MGAT and then TAG by a native DGAT or other activity. Novel DAGproduction could be confirmed and selected for by, for example,performing such a co-transformation in a yeast strain containing lethalSLC1+SLC4 knockouts such as that described by Benghezal et al. (2007;FIG. 2). FIG. 2 of Benghezal et al. (2007) shows that knocking out thetwo yeast LPATS (SLC1 & SLC4) is lethal. The SLC1+SLC4 double yeastmutant can only be maintained because of a complementing plasmid whichprovides one of the sic genes (SLC1 in their case) in trans. Negativeselection by adding FOA to the medium results in the loss of thiscomplementing plasmid (counters election of the Ura selection marker)and renders the cells non viable.

In another embodiment of the invention, the vegetative plant part,transgenic non-human organism or part thereof, preferably a plant orseed, is co-transformed with chimeric DNAs encoding a bifunctionalGPAT/phosphatase and a MGAT which has DGAT activity. This would resultin the production of MAG by the bifunctional GPAT/phosphatase whichwould then be converted to DAG and then TAG by the MGAT.

In a further embodiment, one or more endogenous GPATs with no detectablephosphatase activity are silenced, for example one or more genesencoding GPATs that acylate glycerol-3-phosphate to form LPA in theKennedy Pathway (for example, Arabidopsis GPAT1) is silenced.

In another embodiment, the vegetative plant part, transgenic non-humanorganism or part thereof, preferably a plant or seed, is transformedwith a chimeric DNAs encoding a DGAT1, a DGAT2, a Wrinkled 1 (WRI1)transcription factor, an Oleosin, or a silencing suppressor polypeptide.The chimeric DNAs are preferably covalently linked on one DNA moleculesuch as, for example, a single T-DNA molecule, and the vegetative plantpart, transgenic non-human organism or part thereof is preferablyhomozygous for the one DNA molecule inserted into its genome.

Substrate preferences could be engineered into the novel DAG and TAGsynthesis pathways by, for example, supplying transgenic H1246 yeaststrains expressing MGAT variants with a concentration of a particularfree fatty acid (for example, DHA) that prevents complementation by thewildtype MGAT gene. Only the variants able to use the supplied freefatty acid would grow. Several cycles of MGAT engineering would resultin the production of a MGAT with increased preference for particularfatty acids.

The various Kennedy Pathway complementations and supplementationsdescribed above could be performed in any cell type due to theubiquitous nature of the initial substrate glycerol-3-phosphate. In oneembodiment, the use of transgenes results in increased oil yields.

Polynucleotides

The terms “polynucleotide”, and “nucleic acid” are used interchangeably.They refer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof. Apolynucleotide of the invention may be of genomic, cDNA, semisynthetic,or synthetic origin, double-stranded or single-stranded and by virtue ofits origin or manipulation: (1) is not associated with all or a portionof a polynucleotide with which it is associated in nature, (2) is linkedto a polynucleotide other than that to which it is linked in nature, or(3) does not occur in nature. The following are non-limiting examples ofpolynucleotides: coding or non-coding regions of a gene or genefragment, loci (locus) defined from linkage analysis, exons, introns,messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA),ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, chimeric DNA of any sequence, nucleic acid probes, andprimers. A polynucleotide may comprise modified nucleotides such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization such as by conjugation with a labeling component.

By “isolated polynucleotide” it is meant a polynucleotide which hasgenerally been separated from the polynucleotide sequences with which itis associated or linked in its native state. Preferably, the isolatedpolynucleotide is at least 60% free, more preferably at least 75% free,and more preferably at least 90% free from the polynucleotide sequenceswith which it is naturally associated or linked.

As used herein, the term “gene” is to be taken in its broadest contextand includes the deoxyribonucleotide sequences comprising thetranscribed region and, if translated, the protein coding region, of astructural gene and including sequences located adjacent to the codingregion on both the 5′ and 3′ ends for a distance of at least about 2 kbon either end and which are involved in expression of the gene. In thisregard, the gene includes control signals such as promoters, enhancers,termination and/or polyadenylation signals that are naturally associatedwith a given gene, or heterologous control signals, in which case, thegene is referred to as a “chimeric gene”. The sequences which arelocated 5′ of the protein coding region and which are present on themRNA are referred to as 5′ non-translated sequences. The sequences whichare located 3′ or downstream of the protein coding region and which arepresent on the mRNA are referred to as 3′ non-translated sequences. Theterm “gene” encompasses both cDNA and genomic forms of a gene. A genomicform or clone of a gene contains the coding region which may beinterrupted with non-coding sequences termed “introns”, “interveningregions”, or “intervening sequences.” Introns are segments of a genewhich are transcribed into nuclear RNA (nRNA). Introns may containregulatory elements such as enhancers. Introns are removed or “splicedout” from the nuclear or primary transcript; introns therefore areabsent in the mRNA transcript. The mRNA functions during translation tospecify the sequence or order of amino acids in a nascent polypeptide.The term “gene” includes a synthetic or fusion molecule encoding all orpart of the proteins of the invention described herein and acomplementary nucleotide sequence to any one of the above.

As used herein, “chimeric DNA” refers to any DNA molecule that is notnaturally found in nature; also referred to herein as a “DNA construct”.Typically, chimeric DNA comprises regulatory and transcribed or proteincoding sequences that are not naturally found together in nature.Accordingly, chimeric DNA may comprise regulatory sequences and codingsequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. The openreading frame may or may not be linked to its natural upstream anddownstream regulatory elements. The open reading frame may beincorporated into, for example, the plant genome, in a non-naturallocation, or in a replicon or vector where it is not naturally foundsuch as a bacterial plasmid or a viral vector. The term “chimeric DNA”is not limited to DNA molecules which are replicable in a host, butincludes DNA capable of being ligated into a replicon by, for example,specific adaptor sequences.

A “transgene” is a gene that has been introduced into the genome by atransformation procedure. The term includes a gene in a progeny cell,plant, seed, non-human organism or part thereof which was introducinginto the genome of a progenitor cell thereof. Such progeny cells etc maybe at least a 3^(rd) or 4^(th) generation progeny from the progenitorcell which was the primary transformed cell. Progeny may be produced bysexual reproduction or vegetatively such as, for example, from tubers inpotatoes or ratoons in sugarcane. The term “genetically modified”, andvariations thereof, is a broader term that includes introducing a geneinto a cell by transformation or transduction, mutating a gene in a celland genetically altering or modulating the regulation of a gene in acell, or the progeny of any cell modified as described above.

A “genomic region” as used herein refers to a position within the genomewhere a transgene, or group of transgenes (also referred to herein as acluster), have been inserted into a cell, or predecessor thereof. Suchregions only comprise nucleotides that have been incorporated by theintervention of man such as by methods described herein.

A “recombinant polynucleotide” of the invention refers to a nucleic acidmolecule which has been constructed or modified by artificialrecombinant methods. The recombinant polynucleotide may be present in acell in an altered amount or expressed at an altered rate (e.g., in thecase of mRNA) compared to its native state. In one embodiment, thepolynucleotide is introduced into a cell that does not naturallycomprise the polynucleotide. Typically an exogenous DNA is used as atemplate for transcription of mRNA which is then translated into acontinuous sequence of amino acid residues coding for a polypeptide ofthe invention within the transformed cell. In another embodiment, thepolynucleotide is endogenous to the cell and its expression is alteredby recombinant means, for example, an exogenous control sequence isintroduced upstream of an endogenous gene of interest to enable thetransformed cell to express the polypeptide encoded by the gene.

A recombinant polynucleotide of the invention includes polynucleotideswhich have not been separated from other components of the cell-based orcell-free expression system, in which it is present, and polynucleotidesproduced in said cell-based or cell-free systems which are subsequentlypurified away from at least some other components. The polynucleotidecan be a contiguous stretch of nucleotides existing in nature, orcomprise two or more contiguous stretches of nucleotides from differentsources (naturally occurring and/or synthetic) joined to form a singlepolynucleotide. Typically, such chimeric polynucleotides comprise atleast an open reading frame encoding a polypeptide of the inventionoperably linked to a promoter suitable of driving transcription of theopen reading frame in a cell of interest.

With regard to the defined polynucleotides, it will be appreciated that% identity figures higher than those provided above will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polynucleotide comprises apolynucleotide sequence which is at least 60%, more preferably at least65%, more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, more preferably at least 93%, more preferably at least 94%, morepreferably at least 95%, more preferably at least 96%, more preferablyat least 97%, more preferably at least 98%, more preferably at least99%, more preferably at least 99.1%, more preferably at least 99.2%,more preferably at least 99.3%, more preferably at least 99.4%, morepreferably at least 99.5%, more preferably at least 99.6%, morepreferably at least 99.7%, more preferably at least 99.8%, and even morepreferably at least 99.9% identical to the relevant nominated SEQ ID NO.

A polynucleotide of, or useful for, the present invention mayselectively hybridise, under stringent conditions, to a polynucleotidedefined herein. As used herein, stringent conditions are those that: (1)employ during hybridisation a denaturing agent such as formamide, forexample, 50% (v/v) formamide with 0.1% (w/v) bovine serum albumin, 0.1%Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.; or (2) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 g/ml), 0.1% SDS and 10% dextran sulfateat 42° C. in 0.2×SSC and 0.1% SDS, and/or (3) employ low ionic strengthand high temperature for washing, for example, 0.015 M NaCl/0.0015 Msodium citrate/0.1% SDS at 50° C.

Polynucleotides of the invention may possess, when compared to naturallyoccurring molecules, one or more mutations which are deletions,insertions, or substitutions of nucleotide residues. Polynucleotideswhich have mutations relative to a reference sequence can be eithernaturally occurring (that is to say, isolated from a natural source) orsynthetic (for example, by performing site-directed mutagenesis or DNAshuffling on the nucleic acid as described above).

Polynucleotide for Reducing Expression Levels of Endogenous Proteins

RNA Interference

RNA interference (RNAi) is particularly useful for specificallyinhibiting the production of a particular protein. Although not wishingto be limited by theory, Waterhouse et al. (1998) have provided a modelfor the mechanism by which dsRNA (duplex RNA) can be used to reduceprotein production. This technology relies on the presence of dsRNAmolecules that contain a sequence that is essentially identical to themRNA of the gene of interest or part thereof. Conveniently, the dsRNAcan be produced from a single promoter in a recombinant vector or hostcell, where the sense and anti-sense sequences are flanked by anunrelated sequence which enables the sense and anti-sense sequences tohybridize to form the dsRNA molecule with the unrelated sequence forminga loop structure. The design and production of suitable dsRNA moleculesis well within the capacity of a person skilled in the art, particularlyconsidering Waterhouse et al. (1998), Smith et al. (2000), WO 99/32619,WO 99/53050, WO 99/49029, and WO 01/34815.

In one example, a DNA is introduced that directs the synthesis of an atleast partly double stranded RNA product(s) with homology to the targetgene to be inactivated. The DNA therefore comprises both sense andantisense sequences that, when transcribed into RNA, can hybridize toform the double stranded RNA region. In one embodiment of the invention,the sense and antisense sequences are separated by a spacer region thatcomprises an intron which, when transcribed into RNA, is spliced out.This arrangement has been shown to result in a higher efficiency of genesilencing. The double stranded region may comprise one or two RNAmolecules, transcribed from either one DNA region or two. The presenceof the double stranded molecule is thought to trigger a response from anendogenous system that destroys both the double stranded RNA and alsothe homologous RNA transcript from the target gene, efficiently reducingor eliminating the activity of the target gene.

The length of the sense and antisense sequences that hybridize shouldeach be at least 19 contiguous nucleotides. The full-length sequencecorresponding to the entire gene transcript may be used. The degree ofidentity of the sense and antisense sequences to the targeted transcriptshould be at least 85%, at least 90%, or at least 95-100%. The RNAmolecule may of course comprise unrelated sequences which may functionto stabilize the molecule. The RNA molecule may be expressed under thecontrol of a RNA polymerase II or RNA polymerase III promoter. Examplesof the latter include tRNA or snRNA promoters.

Preferred small interfering RNA (“siRNA”) molecules comprise anucleotide sequence that is identical to about 19-21 contiguousnucleotides of the target mRNA. Preferably, the siRNA sequence commenceswith the dinucleotide AA, comprises a GC-content of about 30-70%(preferably, 30-60%, more preferably 40-60% and more preferably about45%-55%), and does not have a high percentage identity to any nucleotidesequence other than the target in the genome of the organism in which itis to be introduced, for example, as determined by standard BLASTsearch.

microRNA

MicroRNAs (abbreviated miRNAs) are generally 19-25 nucleotides (commonlyabout 20-24 nucleotides in plants) non-coding RNA molecules that arederived from larger precursors that form imperfect stem-loop structures.

miRNAs bind to complementary sequences on target messenger RNAtranscripts (mRNAs), usually resulting in translational repression ortarget degradation and gene silencing.

In plant cells, miRNA precursor molecules are believed to be largelyprocessed in the nucleus. The pri-miRNA (containing one or more localdouble-stranded or “hairpin” regions as well as the usual 5′ “cap” andpolyadenylated tail of an mRNA) is processed to a shorter miRNAprecursor molecule that also includes a stem-loop or fold-back structureand is termed the “pre-miRNA”. In plants, the pre-miRNAs are cleaved bydistinct DICER-like (DCL) enzymes, yielding miRNA:miRNA*duplexes. Priorto transport out of the nucleus, these duplexes are methylated.

In the cytoplasm, the miRNA strand from the miRNA:miRNA duplex isselectively incorporated into an active RNA-induced silencing complex(RISC) for target recognition. The RISC-complexes contain a particularsubset of Argonaute proteins that exert sequence-specific generepression (see, for example, Millar and Waterhouse, 2005; Pasquinelliet al., 2005; Almeida and Allshire, 2005).

Cosuppression

Genes can suppress the expression of related endogenous genes and/ortransgenes already present in the genome, a phenomenon termedhomology-dependent gene silencing. Most of the instances ofhomologydependent gene silencing fall into two classes—those thatfunction at the level of transcription of the transgene, and those thatoperate post-transcriptionally.

Post-transcriptional homology-dependent gene silencing (i.e.,cosuppression) describes the loss of expression of a transgene andrelated endogenous or viral genes in transgenic plants. Cosuppressionoften, but not always, occurs when transgene transcripts are abundant,and it is generally thought to be triggered at the level of mRNAprocessing, localization, and/or degradation. Several models exist toexplain how cosuppression works (see in Taylor, 1997).

One model, the “quantitative” or “RNA threshold” model, proposes thatcells can cope with the accumulation of large amounts of transgenetranscripts, but only up to a point. Once that critical threshold hasbeen crossed, the sequence-dependent degradation of both transgene andrelated endogenous gene transcripts is initiated. It has been proposedthat this mode of cosuppression may be triggered following the synthesisof copy RNA (cRNA) molecules by reverse transcription of the excesstransgene mRNA, presumably by endogenous RNA-dependent RNA polymerases.These cRNAs may hybridize with transgene and endogenous mRNAs, theunusual hybrids targeting homologous transcripts for degradation.However, this model does not account for reports suggesting thatcosuppression can apparently occur in the absence of transgenetranscription and/or without the detectable accumulation of transgenetranscripts.

To account for these data, a second model, the “qualitative” or“aberrant RNA” model, proposes that interactions between transgene RNAand DNA and/or between endogenous and introduced DNAs lead to themethylation of transcribed regions of the genes. The methylated genesare proposed to produce RNAs that are in some way aberrant, theiranomalous features triggering the specific degradation of all relatedtranscripts. Such aberrant RNAs may be produced by complex transgeneloci, particularly those that contain inverted repeats.

A third model proposes that intermolecular base pairing betweentranscripts, rather than cRNA-mRNA hybrids generated through the actionof an RNA-dependent RNA polymerase, may trigger cosuppression. Such basepairing may become more common as transcript levels rise, the putativedouble-stranded regions triggering the targeted degradation ofhomologous transcripts. A similar model proposes intramolecular basepairing instead of intermolecular base pairing between transcripts.

Cosuppression involves introducing an extra copy of a gene or a fragmentthereof into a plant in the sense orientation with respect to a promoterfor its expression. A skilled person would appreciate that the size ofthe sense fragment, its correspondence to target gene regions, and itsdegree of sequence identity to the target gene can vary. In someinstances, the additional copy of the gene sequence interferes with theexpression of the target plant gene. Reference is made to WO 97/20936and EP 0465572 for methods of implementing co-suppression approaches.

Antisense Polynucleotides

The term “antisense polynucleotide” shall be taken to mean a DNA or RNA,or combination thereof, molecule that is complementary to at least aportion of a specific mRNA molecule encoding an endogenous polypeptideand capable of interfering with a post-transcriptional event such asmRNA translation. The use of antisense methods is well known in the art(see for example, G. Hartmann and S. Endres, Manual of AntisenseMethodology, Kluwer (1999)). The use of antisense techniques in plantshas been reviewed by Bourque (1995) and Senior (1998). Bourque (1995)lists a large number of examples of how antisense sequences have beenutilized in plant systems as a method of gene inactivation. Bourque alsostates that attaining 100% inhibition of any enzyme activity may not benecessary as partial inhibition will more than likely result inmeasurable change in the system. Senior (1998) states that antisensemethods are now a very well established technique for manipulating geneexpression.

In one embodiment, the antisense polynucleotide hybridises underphysiological conditions, that is, the antisense polynucleotide (whichis fully or partially single stranded) is at least capable of forming adouble stranded polynucleotide with mRNA encoding a protein such as anendogenous enzyme, for example, DGAT, GPAT, LPAA, LPCAT, PAP, AGPase,under normal conditions in a cell.

Antisense molecules may include sequences that correspond to thestructural genes or for sequences that effect control over the geneexpression or splicing event. For example, the antisense sequence maycorrespond to the targeted coding region of endogenous gene, or the5′-untranslated region (UTR) or the 3′-UTR or combination of these. Itmay be complementary in part to intron sequences, which may be splicedout during or after transcription, preferably only to exon sequences ofthe target gene. In view of the generally greater divergence of theUTRs, targeting these regions provides greater specificity of geneinhibition.

The length of the antisense sequence should be at least 19 contiguousnucleotides, preferably at least 50 nucleotides, and more preferably atleast 100, 200, 500 or 1000 nucleotides. The full-length sequencecomplementary to the entire gene transcript may be used. The length ismost preferably 100-2000 nucleotides. The degree of identity of theantisense sequence to the targeted transcript should be at least 90% andmore preferably 95-100%. The antisense RNA molecule may of coursecomprise unrelated sequences which may function to stabilize themolecule.

Catalytic Polynucleotides

The term “catalytic polynucleotide” refers to a DNA molecule orDNA-containing molecule (also known in the art as a “deoxyribozyme”) oran RNA or RNA-containing molecule (also known as a “ribozyme”) whichspecifically recognizes a distinct substrate and catalyses the chemicalmodification of this substrate. The nucleic acid bases in the catalyticnucleic acid can be bases A, C, G, T (and U for RNA).

Typically, the catalytic nucleic acid contains an antisense sequence forspecific recognition of a target nucleic acid, and a nucleic acidcleaving enzymatic activity (also referred to herein as the “catalyticdomain”). The types of ribozymes that are particularly useful in thisinvention are hammerhead ribozymes (Haseloff and Gerlach, 1988; Perrimanet al., 1992) and hairpin ribozymes (Zolotukhin et al., 1996; Klein etal., 1998; Shippy et al., 1999).

Ribozymes useful in the invention and DNA encoding the ribozymes can bechemically synthesized using methods well known in the art. Theribozymes can also be prepared from a DNA molecule (that upontranscription, yields an RNA molecule) operably linked to an RNApolymerase promoter, for example, the promoter for T7 RNA polymerase orSP6 RNA polymerase. In a separate embodiment, the DNA can be insertedinto an expression cassette or transcription cassette. After synthesis,the RNA molecule can be modified by ligation to a DNA molecule havingthe ability to stabilize the ribozyme and make it resistant to RNase.

As with antisense oligonucleotides, small interfering RNA and microRNAdescribed herein, catalytic polynucleotides useful in the inventionshould be capable of “hybridizing” the target nucleic acid moleculeunder “physiological conditions”, namely those conditions within aplant, algal or fungal cell.

Recombinant Vectors

One embodiment of the present invention includes a recombinant vector,which comprises at least one polynucleotide defined herein and iscapable of delivering the polynucleotide into a host cell. Recombinantvectors include expression vectors. Recombinant vectors containheterologous polynucleotide sequences, that is, polynucleotide sequencesthat are not naturally found adjacent to a polynucleotide definedherein, that preferably, are derived from a different species. Thevector can be either RNA or DNA, either prokaryotic or eukaryotic, andtypically is a viral vector, derived from a virus, or a plasmid. Plasmidvectors typically include additional nucleic acid sequences that providefor easy selection, amplification, and transformation of the expressioncassette in prokaryotic cells, e.g., pUC-derived vectors, pSK-derivedvectors, pGEM-derived vectors, pSP-derived vectors, pBS-derived vectors,or binary vectors containing one or more T-DNA regions. Additionalnucleic acid sequences include origins of replication to provide forautonomous replication of the vector, selectable marker genes,preferably encoding antibiotic or herbicide resistance, unique multiplecloning sites providing for multiple sites to insert nucleic acidsequences or genes encoded in the nucleic acid construct, and sequencesthat enhance transformation of prokaryotic and eukaryotic (especiallyplant) cells.

“Operably linked” as used herein, refers to a functional relationshipbetween two or more nucleic acid (e.g., DNA) segments. Typically, itrefers to the functional relationship of transcriptional regulatoryelement (promoter) to a transcribed sequence. For example, a promoter isoperably linked to a coding sequence of a polynucleotide defined herein,if it stimulates or modulates the transcription of the coding sequencein an appropriate cell. Generally, promoter transcriptional regulatoryelements that are operably linked to a transcribed sequence arephysically contiguous to the transcribed sequence, i.e., they arecis-acting. However, some transcriptional regulatory elements such asenhancers, need not be physically contiguous or located in closeproximity to the coding sequences whose transcription they enhance.

When there are multiple promoters present, each promoter mayindependently be the same or different.

Recombinant vectors may also contain: (a) one or more secretory signalswhich encode signal peptide sequences, to enable an expressedpolypeptide defined herein to be secreted from the cell that producesthe polypeptide, or which provide for localisation of the expressedpolypeptide, for example, for retention of the polypeptide in theendoplasmic reticulum (ER) in the cell, or transfer into a plastid,and/or (b) contain fusion sequences which lead to the expression ofnucleic acid molecules as fusion proteins. Examples of suitable signalsegments include any signal segment capable of directing the secretionor localisation of a polypeptide defined herein. Preferred signalsegments include, but are not limited to, Nicotiana nectarin signalpeptide (U.S. Pat. No. 5,939,288), tobacco extensin signal, or the soyoleosin oil body binding protein signal. Recombinant vectors may alsoinclude intervening and/or untranslated sequences surrounding and/orwithin the nucleic acid sequence of a polynucleotide defined herein.

To facilitate identification of transformants, the recombinant vectordesirably comprises a selectable or screenable marker gene as, or inaddition to, the nucleic acid sequence of a polynucleotide definedherein. By “marker gene” is meant a gene that imparts a distinctphenotype to cells expressing the marker gene and thus, allows suchtransformed cells to be distinguished from cells that do not have themarker. A selectable marker gene confers a trait for which one can“select” based on resistance to a selective agent (e.g., a herbicide,antibiotic, radiation, heat, or other treatment damaging tountransformed cells). A screenable marker gene (or reporter gene)confers a trait that one can identify through observation or testing,that is, by “screening” (e.g., β-glucuronidase, luciferase, GFP or otherenzyme activity not present in untransformed cells). The marker gene andthe nucleotide sequence of interest do not have to be linked, sinceco-transformation of unlinked genes as for example, described in U.S.Pat. No. 4,399,216, is also an efficient process in for example, planttransformation. The actual choice of a marker is not crucial as long asit is functional (i.e., selective) in combination with the cells ofchoice such as a plant cell.

Examples of bacterial selectable markers are markers that conferantibiotic resistance such as ampicillin, erythromycin, chloramphenicol,or tetracycline resistance, preferably kanamycin resistance. Exemplaryselectable markers for selection of plant transformants include, but arenot limited to, a hyg gene which encodes hygromycin B resistance; aneomycin phosphotransferase (nptII) gene conferring resistance tokanamycin, paromomycin, G418; a glutathione-S-transferase gene from ratliver conferring resistance to glutathione derived herbicides as forexample, described in EP 256223; a glutamine synthetase gene conferring,upon overexpression, resistance to glutamine synthetase inhibitors suchas phosphinothricin as for example, described in WO 87/05327; anacetyltransferase gene from Streptomyces viridochromogenes conferringresistance to the selective agent phosphinothricin as for example,described in EP 275957; a gene encoding a 5-enolshikimate-3-phosphatesynthase (EPSPS) conferring tolerance to N-phosphonomethylglycine as forexample, described by Hinchee et al. (1988); a bar gene conferringresistance against bialaphos as for example, described in WO91/02071; anitrilase gene such as bxn from Klebsiella ozaenae which confersresistance to bromoxynil (Stalker et al., 1988); a dihydrofolatereductase (DHFR) gene conferring resistance to methotrexate (Thillet etal., 1988); a mutant acetolactate synthase gene (ALS) which confersresistance to imidazolinone, sulfonylurea, or other ALS-inhibitingchemicals (EP 154,204); a mutated anthranilate synthase gene thatconfers resistance to 5-methyl tryptophan; or a dalapon dehalogenasegene that confers resistance to the herbicide.

Preferred screenable markers include, but are not limited to, a uidAgene encoding a β-glucuronidase (GUS) enzyme for which variouschromogenic substrates are known; a β-galactosidase gene encoding anenzyme for which chromogenic substrates are known; an aequorin gene(Prasher et al., 1985) which may be employed in calcium-sensitivebioluminescence detection; a green fluorescent protein gene (Niedz etal., 1995) or derivatives thereof; or a luciferase (luc) gene (Ow etal., 1986) which allows for bioluminescence detection. By “reportermolecule” it is meant a molecule that, by its chemical nature, providesan analytically identifiable signal that facilitates determination ofpromoter activity by reference to protein product.

Preferably, the recombinant vector is stably incorporated into thegenome of the cell such as the plant cell. Accordingly, the recombinantvector may comprise appropriate elements which allow the vector to beincorporated into the genome, or into a chromosome of the cell.

Expression Vector

As used herein, an “expression vector” is a DNA or RNA vector that iscapable of transforming a host cell and of effecting expression of oneor more specified polynucleotides. Preferably, the expression vector isalso capable of replicating within the host cell. Expression vectors canbe either prokaryotic or eukaryotic, and are typically viruses orplasmids. Expression vectors of the present invention include anyvectors that function (i.e., direct gene expression) in host cells ofthe present invention, including in bacterial, fungal, endoparasite,arthropod, animal, algal, and plant cells. Particularly preferredexpression vectors of the present invention can direct gene expressionin yeast, algae and/or plant cells.

Expression vectors of the present invention contain regulatory sequencessuch as transcription control sequences, translation control sequences,origins of replication, and other regulatory sequences that arecompatible with the host cell and that control the expression ofpolynucleotides of the present invention. In particular, expressionvectors of the present invention include transcription controlsequences. Transcription control sequences are sequences which controlthe initiation, elongation, and termination of transcription.Particularly important transcription control sequences are those whichcontrol transcription initiation such as promoter, enhancer, operatorand repressor sequences. Suitable transcription control sequencesinclude any transcription control sequence that can function in at leastone of the recombinant cells of the present invention. The choice of theregulatory sequences used depends on the target organism such as a plantand/or target organ or tissue of interest. Such regulatory sequences maybe obtained from any eukaryotic organism such as plants or plantviruses, or may be chemically synthesized. A variety of suchtranscription control sequences are known to those skilled in the art.Particularly preferred transcription control sequences are promotersactive in directing transcription in plants, either constitutively orstage and/or tissue specific, depending on the use of the plant orpart(s) thereof.

A number of vectors suitable for stable transfection of plant cells orfor the establishment of transgenic plants have been described in forexample, Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985,supp. 1987, Weissbach and Weissbach, Methods for Plant MolecularBiology, Academic Press, 1989, and Gelvin et al., Plant MolecularBiology Manual, Kluwer Academic Publishers, 1990. Typically, plantexpression vectors include for example, one or more cloned plant genesunder the transcriptional control of 5′ and 3′ regulatory sequences anda dominant selectable marker. Such plant expression vectors also cancontain a promoter regulatory region (e.g., a regulatory regioncontrolling inducible or constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific expression), atranscription initiation start site, a ribosome binding site, an RNAprocessing signal, a transcription termination site, and/or apolyadenylation signal.

A number of constitutive promoters that are active in plant cells havebeen described. Suitable promoters for constitutive expression in plantsinclude, but are not limited to, the cauliflower mosaic virus (CaMV) 35Spromoter, the Figwort mosaic virus (FMV) 35S, the sugarcane bacilliformvirus promoter, the commelina yellow mottle virus promoter, thelight-inducible promoter from the small subunit of theribulose-1,5-bis-phosphate carboxylase, the rice cytosolictriosephosphate isomerase promoter, the adeninephosphoribosyltransferase promoter of Arabidopsis, the rice actin 1 genepromoter, the mannopine synthase and octopine synthase promoters, theAdh promoter, the sucrose synthase promoter, the R gene complexpromoter, and the chlorophyll α/β binding protein gene promoter. Thesepromoters have been used to create DNA vectors that have been expressedin plants, see for example, WO 84/02913. All of these promoters havebeen used to create various types of plant-expressible recombinant DNAvectors.

For the purpose of expression in source tissues of the plant such as theleaf, seed, root or stem, it is preferred that the promoters utilized inthe present invention have relatively high expression in these specifictissues. For this purpose, one may choose from a number of promoters forgenes with tissue- or cell-specific, or -enhanced expression. Examplesof such promoters reported in the literature include, the chloroplastglutamine synthetase GS2 promoter from pea, the chloroplastfructose-1,6-biphosphatase promoter from wheat, the nuclearphotosynthetic ST-LS1 promoter from potato, the serine/threonine kinasepromoter and the glucoamylase (CHS) promoter from Arabidopsis thaliana.Also reported to be active in photosynthetically active tissues are theribulose-1,5-bisphosphate carboxylase promoter from eastern larch (Larixlaricina), the promoter for the Cab gene, Cab6, from pine, the promoterfor the Cab-1 gene from wheat, the promoter for the Cab-1 gene fromspinach, the promoter for the Cab 1R gene from rice, the pyruvate,orthophosphate dikinase (PPDK) promoter from Zea mays, the promoter forthe tobacco Lhcb1*2 gene, the Arabidopsis thaliana Suc2 sucrose-H³⁰symporter promoter, and the promoter for the thylakoid membrane proteingenes from spinach (PsaD, PsaF, PsaE, PC, FNR, AtpC, AtpD, Cab, RbcS).Other promoters for the chlorophyll α/β-binding proteins may also beutilized in the present invention such as the promoters for LhcB geneand PsbP gene from white mustard (Sinapis alba).

A variety of plant gene promoters that are regulated in response toenvironmental, hormonal, chemical, and/or developmental signals, alsocan be used for expression of RNA-binding protein genes in plant cells,including promoters regulated by (1) heat, (2) light (e.g., pea RbcS-3Apromoter, maize RbcS promoter), (3) hormones such as abscisic acid, (4)wounding (e.g., WunI), or (5) chemicals such as methyl jasmonate,salicylic acid, steroid hormones, alcohol, Safeners (WO 97/06269), or itmay also be advantageous to employ (6) organ-specific promoters.

As used herein, the term “plant storage organ specific promoter” refersto a promoter that preferentially, when compared to other plant tissues,directs gene transcription in a storage organ of a plant. Preferably,the promoter only directs expression of a gene of interest in thestorage organ, and/or expression of the gene of interest in other partsof the plant such as leaves is not detectable by Northern blot analysisand/or RT-PCR. Typically, the promoter drives expression of genes duringgrowth and development of the storage organ, in particular during thephase of synthesis and accumulation of storage compounds in the storageorgan. Such promoters may drive gene expression in the entire plantstorage organ or only part thereof such as the seedcoat, embryo orcotyledon(s) in seeds of dicotyledonous plants or the endosperm oraleurone layer of seeds of monocotyledonous plants.

For the purpose of expression in sink tissues of the plant such as thetuber of the potato plant, the fruit of tomato, or the seed of soybean,canola, cotton, Zea mays, wheat, rice, and barley, it is preferred thatthe promoters utilized in the present invention have relatively highexpression in these specific tissues. A number of promoters for geneswith tuber-specific or -enhanced expression are known, including theclass I patatin promoter, the promoter for the potato tuber ADPGPPgenes, both the large and small subunits, the sucrose synthase promoter,the promoter for the major tuber proteins, including the 22 kD proteincomplexes and proteinase inhibitors, the promoter for the granule boundstarch synthase gene (GBSS), and other class I and II patatinspromoters. Other promoters can also be used to express a protein inspecific tissues such as seeds or fruits. The promoter for β-conglycininor other seed-specific promoters such as the napin, zein, linin andphaseolin promoters, can be used. Root specific promoters may also beused. An example of such a promoter is the promoter for the acidchitinase gene. Expression in root tissue could also be accomplished byutilizing the root specific subdomains of the CaMV 35S promoter thathave been identified.

In a particularly preferred embodiment, the promoter directs expressionin tissues and organs in which lipid biosynthesis take place. Suchpromoters may act in seed development at a suitable time for modifyinglipid composition in seeds.

In an embodiment, the promoter is a plant storage organ specificpromoter. In one embodiment, the plant storage organ specific promoteris a seed specific promoter. In a more preferred embodiment, thepromoter preferentially directs expression in the cotyledons of adicotyledonous plant or in the endosperm of a monocotyledonous plant,relative to expression in the embryo of the seed or relative to otherorgans in the plant such as leaves. Preferred promoters forseed-specific expression include: 1) promoters from genes encodingenzymes involved in lipid biosynthesis and accumulation in seeds such asdesaturases and elongases, 2) promoters from genes encoding seed storageproteins, and 3) promoters from genes encoding enzymes involved incarbohydrate biosynthesis and accumulation in seeds. Seed specificpromoters which are suitable are, the oilseed rape napin gene promoter(U.S. Pat. No. 5,608,152), the Vicia faba USP promoter (Baumlein et al.,1991), the Arabidopsis oleosin promoter (WO 98/45461), the Phaseolusvulgaris phaseolin promoter (U.S. Pat. No. 5,504,200), the Brassica Bce4promoter (WO 91/13980), or the legumin B4 promoter (Baumlein et al.,1992), and promoters which lead to the seed-specific expression inmonocots such as maize, barley, wheat, rye, rice and the like. Notablepromoters which are suitable are the barley lpt2 or lpt1 gene promoter(WO 95/15389 and WO 95/23230), or the promoters described in WO 99/16890(promoters from the barley hordein gene, the rice glutelin gene, therice oryzin gene, the rice prolamin gene, the wheat gliadin gene, thewheat glutelin gene, the maize zein gene, the oat glutelin gene, thesorghum kasirin gene, the rye secalin gene). Other promoters includethose described by Broun et al. (1998), Potenza et al. (2004), US20070192902 and US 20030159173. In an embodiment, the seed specificpromoter is preferentially expressed in defined parts of the seed suchas the cotyledon(s) or the endosperm. Examples of cotyledon specificpromoters include, but are not limited to, the FP1 promoter (Ellerstromet al., 1996), the pea legumin promoter (Perrin et al., 2000), and thebean phytohemagglutnin promoter (Perrin et al., 2000). Examples ofendosperm specific promoters include, but are not limited to, the maizezein-1 promoter (Chikwamba et al., 2003), the rice glutelin-1 promoter(Yang et al., 2003), the barley D-hordein promoter (Horvath et al.,2000) and wheat HMW glutenin promoters (Alvarez et al., 2000). In afurther embodiment, the seed specific promoter is not expressed, or isonly expressed at a low level, in the embryo and/or after the seedgerminates.

In another embodiment, the plant storage organ specific promoter is atuber specific promoter. Examples include, but are not limited to, thepotato patatin B33, PAT21 and GBSS promoters, as well as the sweetpotato sporamin promoter (for review, see Potenza et al., 2004). In apreferred embodiment, the promoter directs expression preferentially inthe pith of the tuber, relative to the outer layers (skin, bark) or theembryo of the tuber.

In another embodiment, the plant storage organ specific promoter is afruit specific promoter. Examples include, but are not limited to, thetomato polygalacturonase, E8 and Pds promoters, as well as the apple ACCoxidase promoter (for review, see Potenza et al., 2004). In a preferredembodiment, the promoter preferentially directs expression in the edibleparts of the fruit, for example the pith of the fruit, relative to theskin of the fruit or the seeds within the fruit.

In an embodiment, the inducible promoter is the Aspergillus nidulans alcsystem. Examples of inducible expression systems which can be usedinstead of the Aspergillus nidulans alc system are described in a reviewby Padidam (2003) and Corrado and Karali (2009). These includetetracycline repressor (TetR)-based and tetracycline inducible systems(Gatz, 1997), tetracycline repressor-based andtetracycline-inactivatable systems (Weinmann et al., 1994),glucocorticoid receptor-based (Picard, 1994), estrogen receptor-basedand other steroid-inducible systems systems (Bruce et al., 2000),glucocorticoid receptor-, tetracycline repressor-based dual controlsystems (Bohner et al., 1999), ecdysone receptor-based,insecticide-inducible systems (Martinez et al., 1999, Padidam et al.,2003, Unger et al, 2002, Riddiford et al., 2000, Dhadialla et al., 1998,Martinez and Jepson, 1999), AlcR-based, ethanol-inducible systems(Felenbok, 1991) and ACEI-based, copper-inducible systems (Mett et al.,1993).

In another embodiment, the inducible promoter is a safener induciblepromoter such as, for example, the maize ln2-1 or ln2-2 promoter(Hershey and Stoner, 1991), the safener inducible promoter is the maizeGST-27 promoter (Jepson et al., 1994), or the soybean GH2/4 promoter(Ulmasov et al., 1995).

Safeners are a group of structurally diverse chemicals used to increasethe plant's tolerance to the toxic effects of an herbicidal compound.Examples of these compounds include naphthalic anhydride andN,N-diallyl-2,2-dichloroacetamide (DDCA), which protect maize andsorghum against thiocarbamate herbicides; cyometrinil, which protectssorghum against metochlor; triapenthenol, which protects soybeansagainst metribuzin; and substituted benzenesulfonamides, which improvethe tolerance of several cereal crop species to sulfonylurea herbicides.

In another embodiment, the inducible promoter is a senescence induciblepromoter such as, for example, senescence-inducible promoter SAG(senescence associated gene) 12 and SAG 13 from Arabidopsis (Gan, 1995;Gan and Amasino, 1995) and LSC54 from Brassica napus(Buchanan-Wollaston, 1994).

For expression in vegetative tissue leaf-specific promoters, such as theribulose biphosphate carboxylase (RBCS) promoters, can be used. Forexample, the tomato RBCS1, RBCS2 and RBCS3A genes are expressed inleaves and light grown seedlings (Meier et al., 1997). A ribulosebisphosphate carboxylase promoters expressed almost exclusively inmesophyll cells in leaf blades and leaf sheaths at high levels,described by Matsuoka et al. (1994), can be used. Another leaf-specificpromoter is the light harvesting chlorophyll a/b binding protein genepromoter (see, Shiina et al., 1997). The Arabidopsis thalianamyb-related gene promoter (Atmyb5) described by Li et al. (1996), isleaf-specific. The Atmyb5 promoter is expressed in developing leaftrichomes, stipules, and epidermal cells on the margins of young rosetteand cauline leaves, and in immature seeds. A leaf promoter identified inmaize by Busk et al. (1997), can also be used.

In some instances, for example when LEC2 or BBM is recombinantlyexpressed, it may be desirable that the transgene is not expressed athigh levels. An example of a promoter which can be used in suchcircumstances is a truncated napin A promoter which retains theseed-specific expression pattern but with a reduced expression level(Tan et al., 2011).

The 5′ non-translated leader sequence can be derived from the promoterselected to express the heterologous gene sequence of the polynucleotideof the present invention, or may be heterologous with respect to thecoding region of the enzyme to be produced, and can be specificallymodified if desired so as to increase translation of mRNA. For a reviewof optimizing expression of transgenes, see Koziel et al. (1996). The 5′non-translated regions can also be obtained from plant viral RNAs(Tobacco mosaic virus, Tobacco etch virus, Maize dwarf mosaic virus,Alfalfa mosaic virus, among others) from suitable eukaryotic genes,plant genes (wheat and maize chlorophyll a/b binding protein geneleader), or from a synthetic gene sequence. The present invention is notlimited to constructs wherein the non-translated region is derived fromthe 5′ non-translated sequence that accompanies the promoter sequence.The leader sequence could also be derived from an unrelated promoter orcoding sequence. Leader sequences useful in context of the presentinvention comprise the maize Hsp70 leader (U.S. Pat. No. 5,362,865 andU.S. Pat. No. 5,859,347), and the TMV omega element.

The termination of transcription is accomplished by a 3′ non-translatedDNA sequence operably linked in the expression vector to thepolynucleotide of interest. The 3′ non-translated region of arecombinant DNA molecule contains a polyadenylation signal thatfunctions in plants to cause the addition of adenylate nucleotides tothe 3′ end of the RNA. The 3′ non-translated region can be obtained fromvarious genes that are expressed in plant cells. The nopaline synthase3′ untranslated region, the 3′ untranslated region from pea smallsubunit Rubisco gene, the 3′ untranslated region from soybean 7S seedstorage protein gene are commonly used in this capacity. The 3′transcribed, non-translated regions containing the polyadenylate signalof Agrobacterium tumor-inducing (Ti) plasmid genes are also suitable.

Recombinant DNA technologies can be used to improve expression of atransformed polynucleotide by manipulating for example, the number ofcopies of the polynucleotide within a host cell, the efficiency withwhich those polynucleotide are transcribed, the efficiency with whichthe resultant transcripts are translated, and the efficiency ofpost-translational modifications. Recombinant techniques useful forincreasing the expression of polynucleotides defined herein include, butare not limited to, operatively linking the polynucleotide to ahigh-copy number plasmid, integration of the polynucleotide moleculeinto one or more host cell chromosomes, addition of vector stabilitysequences to the plasmid, substitutions or modifications oftranscription control signals (e.g., promoters, operators, enhancers),substitutions or modifications of translational control signals (e.g.,ribosome binding sites, Shine-Dalgarno sequences), modification of thepolynucleotide to correspond to the codon usage of the host cell, andthe deletion of sequences that destabilize transcripts.

Transfer Nucleic Acids

Transfer nucleic acids can be used to deliver an exogenouspolynucleotide to a cell and comprise one, preferably two, bordersequences and a polynucleotide of interest. The transfer nucleic acidmay or may not encode a selectable marker. Preferably, the transfernucleic acid forms part of a binary vector in a bacterium, where thebinary vector further comprises elements which allow replication of thevector in the bacterium, selection, or maintenance of bacterial cellscontaining the binary vector. Upon transfer to a eukaryotic cell, thetransfer nucleic acid component of the binary vector is capable ofintegration into the genome of the eukaryotic cell.

As used herein, the term “extrachromosomal transfer nucleic acid” refersto a nucleic acid molecule that is capable of being transferred from abacterium such as Agrobacterium sp., to a eukaryotic cell such as aplant leaf cell. An extrachromosomal transfer nucleic acid is a geneticelement that is well-known as an element capable of being transferred,with the subsequent integration of a nucleotide sequence containedwithin its borders into the genome of the recipient cell. In thisrespect, a transfer nucleic acid is flanked, typically, by two “border”sequences, although in some instances a single border at one end can beused and the second end of the transferred nucleic acid is generatedrandomly in the transfer process. A polynucleotide of interest istypically positioned between the left border-like sequence and the rightborder-like sequence of a transfer nucleic acid. The polynucleotidecontained within the transfer nucleic acid may be operably linked to avariety of different promoter and terminator regulatory elements thatfacilitate its expression, that is, transcription and/or translation ofthe polynucleotide. Transfer DNAs (T-DNAs) from Agrobacterium sp. suchas Agrobacterium tumefaciens or Agrobacterium rhizogenes, and man madevariants/mutants thereof are probably the best characterized examples oftransfer nucleic acids. Another example is P-DNA (“plant-DNA”) whichcomprises T-DNA border-like sequences from plants.

As used herein, “T-DNA” refers to for example, T-DNA of an Agrobacteriumtumefaciens Ti plasmid or from an Agrobacterium rhizogenes Ri plasmid,or man made variants thereof which function as T-DNA. The T-DNA maycomprise an entire T-DNA including both right and left border sequences,but need only comprise the minimal sequences required in cis fortransfer, that is, the right and T-DNA border sequence. The T-DNAs ofthe invention have inserted into them, anywhere between the right andleft border sequences (if present), the polynucleotide of interestflanked by target sites for a site-specific recombinase. The sequencesencoding factors required in trans for transfer of the T-DNA into aplant cell such as vir genes, may be inserted into the T-DNA, or may bepresent on the same replicon as the T-DNA, or preferably are in trans ona compatible replicon in the Agrobacterium host. Such “binary vectorsystems” are well known in the art.

As used herein, “P-DNA” refers to a transfer nucleic acid isolated froma plant genome, or man made variants/mutants thereof, and comprises ateach end, or at only one end, a T-DNA border-like sequence. Theborder-like sequence preferably shares at least 50%, at least 60%, atleast 70%, at least 75%, at least 80%, at least 90% or at least 95%, butless than 100% sequence identity, with a T-DNA border sequence from anAgrobacterium sp. such as Agrobacterium tumefaciens or Agrobacteriumrhizogenes. Thus, P-DNAs can be used instead of T-DNAs to transfer anucleotide sequence contained within the P-DNA from, for exampleAgrobacterium, to another cell. The P-DNA, before insertion of theexogenous polynucleotide which is to be transferred, may be modified tofacilitate cloning and should preferably not encode any proteins. TheP-DNA is characterized in that it contains, at least a right bordersequence and preferably also a left border sequence.

As used herein, a “border” sequence of a transfer nucleic acid can beisolated from a selected organism such as a plant or bacterium, or be aman made variant/mutant thereof. The border sequence promotes andfacilitates the transfer of the polynucleotide to which it is linked andmay facilitate its integration in the recipient cell genome. In anembodiment, a border-sequence is between 5-100 base pairs (bp) inlength, 10-80 bp in length, 15-75 bp in length, 15-60 bp in length,15-50 bp in length, 15-40 bp in length, 15-30 bp in length, 16-30 bp inlength, 20-30 bp in length, 21-30 bp in length, 22-30 bp in length,23-30 bp in length, 24-30 bp in length, 25-30 bp in length, or 26-30 bpin length. Border sequences from T-DNA from Agrobacterium sp. are wellknown in the art and include those described in Lacroix et al. (2008),Tzfira and Citovsky (2006) and Glevin (2003).

Whilst traditionally only Agrobacterium sp. have been used to transfergenes to plants cells, there are now a large number of systems whichhave been identified/developed which act in a similar manner toAgrobacterium sp. Several non-Agrobacterium species have recently beengenetically modified to be competent for gene transfer (Chung et al.,2006; Broothaerts et al., 2005). These include Rhizobium sp. NGR234,Sinorhizobium meliloti and Mezorhizobium loti. The bacteria are madecompetent for gene transfer by providing the bacteria with the machineryneeded for the transformation process, that is, a set of virulence genesencoded by an Agrobacterium Ti-plasmid and the T-DNA segment residing ona separate, small binary plasmid. Bacteria engineered in this way arecapable of transforming different plant tissues (leaf disks, calli andoval tissue), monocots or dicots, and various different plant species(e.g., tobacco, rice).

Direct transfer of eukaryotic expression plasmids from bacteria toeukaryotic hosts was first achieved several decades ago by the fusion ofmammalian cells and protoplasts of plasmid-carrying Escherichia coli(Schaffner, 1980). Since then, the number of bacteria capable ofdelivering genes into mammalian cells has steadily increased (Weiss,2003), being discovered by four groups independently (Sizemore et al.1995; Courvalin et al., 1995; Powell et al., 1996; Darji et al., 1997).

Attenuated Shigella flexneri, Salmonella typhimurium or E. coli that hadbeen rendered invasive by the virulence plasmid (pWR100) of S. flexnerihave been shown to be able to transfer expression plasmids afterinvasion of host cells and intracellular death due to metabolicattenuation. Mucosal application, either nasally or orally, of suchrecombinant Shigella or Salmonella induced immune responses against theantigen that was encoded by the expression plasmids. In the meantime,the list of bacteria that was shown to be able to transfer expressionplasmids to mammalian host cells in vitro and in vivo has been more thendoubled and has been documented for S. typhi, S. choleraesuis, Listeriamonocytogenes, Yersinia pseudotuberculosis, and Y. enterocolitica(Fennelly et al., 1999; Shiau et al., 2001; Dietrich et al., 1998; Henseet al., 2001; Al-Marini et al., 2002).

In general, it could be assumed that all bacteria that are able to enterthe cytosol of the host cell (like S. flexneri or L. monocytogenes) andlyse within this cellular compartment, should be able to transfer DNA.This is known as ‘abortive’ or ‘suicidal’ invasion as the bacteria haveto lyse for the DNA transfer to occur (Grillot-Courvalin et al., 1999).In addition, even many of the bacteria that remain in the phagocyticvacuole (like S. typhimurium) may also be able to do so. Thus,recombinant laboratory strains of E. coli that have been engineered tobe invasive but are unable of phagosomal escape, could deliver theirplasmid load to the nucleus of the infected mammalian cell nevertheless(Grillot-Courvalin et al., 1998). Furthermore, Agrobacterium tumefacienshas recently also been shown to introduce transgenes into mammaliancells (Kunik et al., 2001).

As used herein, the terms “transfection”, “transformation” andvariations thereof are generally used interchangeably. “Transfected” or“transformed” cells may have been manipulated to introduce thepolynucleotide(s) of interest, or may be progeny cells derivedtherefrom.

Recombinant Cells

The invention also provides a recombinant cell, for example, arecombinant plant cell, which is a host cell transformed with one ormore polynucleotides or vectors defined herein, or combination thereof.The term “recombinant cell” is used interchangeably with the term“transgenic cell” herein. Suitable cells of the invention include anycell that can be transformed with a polynucleotide or recombinant vectorof the invention, encoding for example, a polypeptide or enzymedescribed herein. The cell is preferably a cell which is thereby capableof being used for producing lipid. The recombinant cell may be a cell inculture, a cell in vitro, or in an organism such as for example, aplant, or in an organ such as, for example, a seed or a leaf.Preferably, the cell is in a plant, more preferably in the seed of aplant. In one embodiment, the recombinant cell is a non-human cell.

Host cells into which the polynucleotide(s) are introduced can be eitheruntransformed cells or cells that are already transformed with at leastone nucleic acid. Such nucleic acids may be related to lipid synthesis,or unrelated. Host cells of the present invention either can beendogenously (i.e., naturally) capable of producing polypeptide(s)defined herein, in which case the recombinant cell derived therefrom hasan enhanced capability of producing the polypeptide(s), or can becapable of producing said polypeptide(s) only after being transformedwith at least one polynucleotide of the invention. In an embodiment, arecombinant cell of the invention has an enhanced capacity to producenon-polar lipid.

Host cells of the present invention can be any cell capable of producingat least one protein described herein, and include bacterial, fungal(including yeast), parasite, arthropod, animal, algal, and plant cells.The cells may be prokaryotic or eukaryotic. Preferred host cells areyeast, algal and plant cells. In a preferred embodiment, the plant cellis a seed cell, in particular, a cell in a cotyledon or endosperm of aseed. In one embodiment, the cell is an animal cell. The animal cell maybe of any type of animal such as, for example, a non-human animal cell,a non-human vertebrate cell, a non-human mammalian cell, or cells ofaquatic animals such as fish or crustacea, invertebrates, insects, etc.Non limiting examples of arthropod cells include insect cells such asSpodoptera frugiperda (Sf) cells, for example, Sf9, Sf21, Trichoplusiani cells, and Drosophila S2 cells. An example of a bacterial cell usefulas a host cell of the present invention is Synechococcus spp. (alsoknown as Synechocystis spp.), for example Synechococcus elongatus.Examples of algal cells useful as host cells of the present inventioninclude, for example, Chlamydomonas sp. (for example, Chlamydomonasreinhardtii), Dunaliella sp., Haematococcus sp., Chlorella sp.,Thraustochytrium sp., Schizochytrium sp., and Volvox sp.

Host cells for expression of the instant nucleic acids may includemicrobial hosts that grow on a variety of feedstocks, including simpleor complex carbohydrates, organic acids and alcohols and/or hydrocarbonsover a wide range of temperature and pH values. Preferred microbialhosts are oleaginous organisms that are naturally capable of non-polarlipid synthesis.

The host cells may be of an organism suitable for a fermentationprocess, such as, for example, Yarrowia lipolytica or other yeasts.

Transgenic Plants

The invention also provides a plant comprising an exogenouspolynucleotide or polypeptide of the invention, a cell of the invention,a vector of the invention, or a combination thereof. The term “plant”refers to whole plants, whilst the term “part thereof” refers to plantorgans (e.g., leaves, stems, roots, flowers, fruit), single cells (e.g.,pollen), seed, seed parts such as an embryo, endosperm, scutellum orseed coat, plant tissue such as vascular tissue, plant cells and progenyof the same. As used herein, plant parts comprise plant cells.

As used herein, the term “plant” is used in it broadest sense. Itincludes, but is not limited to, any species of grass, ornamental ordecorative plant, crop or cereal (e.g., oilseed, maize, soybean), fodderor forage, fruit or vegetable plant, herb plant, woody plant, flowerplant, or tree. It is not meant to limit a plant to any particularstructure. It also refers to a unicellular plant (e.g., microalga). Theterm “part thereof” in reference to a plant refers to a plant cell andprogeny of same, a plurality of plant cells that are largelydifferentiated into a colony (e.g., volvox), a structure that is presentat any stage of a plant's development, or a plant tissue. Suchstructures include, but are not limited to, leaves, stems, flowers,fruits, nuts, roots, seed, seed coat, embryos. The term “plant tissue”includes differentiated and undifferentiated tissues of plants includingthose present in leaves, stems, flowers, fruits, nuts, roots, seed, forexample, embryonic tissue, endosperm, dermal tissue (e.g., epidermis,periderm), vascular tissue (e.g., xylem, phloem), or ground tissue(comprising parenchyma, collenchyma, and/or sclerenchyma cells), as wellas cells in culture (e.g., single cells, protoplasts, callus, embryos,etc.). Plant tissue may be in planta, in organ culture, tissue culture,or cell culture.

A “transgenic plant”, “genetically modified plant” or variations thereofrefers to a plant that contains a transgene not found in a wild-typeplant of the same species, variety or cultivar. Transgenic plants asdefined in the context of the present invention include plants and theirprogeny which have been genetically modified using recombinanttechniques to cause production of at least one polypeptide definedherein in the desired plant or part thereof. Transgenic plant parts hasa corresponding meaning.

The terms “seed” and “grain” are used interchangeably herein. “Grain”refers to mature grain such as harvested grain or grain which is stillon a plant but ready for harvesting, but can also refer to grain afterimbibition or germination, according to the context. Mature graincommonly has a moisture content of less than about 18-20%. In a pretendembodiment, the moisture content of the grain is at a level which isgenerally regarded as safe for storage, preferably between 5% and 15%,between 6% and 8%, between 8% and 10%, or between 12% and 15%.“Developing seed” as used herein refers to a seed prior to maturity,typically found in the reproductive structures of the plant afterfertilisation or anthesis, but can also refer to such seeds prior tomaturity which are isolated from a plant. Mature seed commonly has amoisture content of less than about 18-20%. In a preferred embodiment,the moisture content of the seed is at a level which is generallyregarded as safe for storage, preferably between 5% and 15%, between 6%and 8%, between 8% and 10%, or between 12% and 15%.

As used herein, the term “plant storage organ” refers to a part of aplant specialized to store energy in the form of for example, proteins,carbohydrates, lipid. Examples of plant storage organs are seed, fruit,tuberous roots, and tubers. A preferred plant storage organ of theinvention is seed.

As used herein, the term “phenotypically normal” refers to a geneticallymodified plant or part thereof, particularly a storage organ such as aseed, tuber or fruit of the invention not having a significantly reducedability to grow and reproduce when compared to an unmodified plant orplant thereof. In an embodiment, the genetically modified plant or partthereof which is phenotypically normal comprises a recombinantpolynucleotide encoding a silencing suppressor operably linked to aplant storage organ specific promoter and has an ability to grow orreproduce which is essentially the same as a corresponding plant or partthereof not comprising said polynucleotide. Preferably, the biomass,growth rate, germination rate, storage organ size, seed size and/or thenumber of viable seeds produced is not less than 90% of that of a plantlacking said recombinant polynucleotide when grown under identicalconditions. This term does not encompass features of the plant which maybe different to the wild-type plant but which do not effect theusefulness of the plant for commercial purposes such as, for example, aballerina phenotype of seedling leaves.

Plants provided by or contemplated for use in the practice of thepresent invention include both monocotyledons and dicotyledons. Inpreferred embodiments, the plants of the present invention are cropplants (for example, cereals and pulses, maize, wheat, potatoes,tapioca, rice, sorghum, millet, cassava, barley, or pea), or otherlegumes. The plants may be grown for production of edible roots, tubers,leaves, stems, flowers or fruit. The plants may be vegetable orornamental plants. The plants of the invention may be: Acrocomiaaculeata (macauba palm), Arabidopsis thaliana, Aracinis hypogaea(peanut), Astrocaryum murumuru (murumuru), Astrocaryum vulgare (tucumã),Attalea geraensis (Indaiá-rateiro), Attalea humilis (American oil palm),Attalea oleifera (andaiá), Attalea phalerata (uricuri), Attalea speciosa(babassu), Avena sativa (oats), Beta vulgaris (sugar beet), Brassica sp.such as Brassica carinata, Brassica juncea, Brassica napobrassica,Brassica napus (canola), Camelina sativa (false flax), Cannabis sativa(hemp), Carthamus tinctorius (safflower), Caryocar brasiliense (pequi),Cocos nucifera (Coconut), Crambe abyssinica (Abyssinian kale), Cucumismelo (melon), Elaeis guineensis (African palm), Glycine max (soybean),Gossypium hirsutum (cotton), Helianthus sp. such as Helianthus animus(sunflower), Hordeum vulgare (barley), Jatropha curcas (physic nut),Joannesia princeps (arara nut-tree), Lemna sp. (duckweed) such as Lemnaaequinoctialis, Lemna disperma, Lemna ecuadoriensis, Lemna gibba(swollen duckweed), Lemna japonica, Lemna minor, Lemna minuta, Lemnaobscura, Lemna paucicostata, Lemna perpusilla, Lemna tenera, Lemnatrisulca, Lemna turionifera, Lemna valdiviana, Lemna yungensis, Licaniarigida (oiticica), Linum usitatissimum (flax), Lupinus angustifolius(lupin), Mauritia flexuosa (buriti palm), Maximiliana maripa (inajapalm), Miscanthus sp. such as Miscanthus×giganteus and Miscanthussinensis, Nicotiana sp. (tabacco) such as Nicotiana tabacum or Nicotianabenthamiana, Oenocarpus bacaba (bacaba-do-azeite), Oenocarpus bataua(patauã), Oenocarpus distichus (bacaba-de-leque), Oryza sp. (rice) suchas Oryza sativa and Oryza glaberrima, Panicum virgatum (switchgrass),Paraqueiba paraensis (mari), Persea amencana (avocado), Pongamia pinnata(Indian beech), Populus trichocarpa, Ricinus communis (castor),Saccharum sp. (sugarcane), Sesamum indicum (sesame), Solanum tuberosum(potato), Sorghum sp. such as Sorghum bicolor, Sorghum vulgare,Theobroma grandiforum (cupuassu), Trifolium sp., Trithrinax brasiliensis(Brazilian needle palm), Triticum sp. (wheat) such as Triticum aestivum,Zea mays (corn), alfalfa (Medicago sativa), rye (Secale cerale), sweetpotato (Lopmoea batatus), cassava (Manihot esculenta), coffee (Cofeaspp.), pineapple (Anana comosus), citris tree (Citrus spp.), cocoa(Theobroma cacao), tea (Camellia senensis), banana (Musa spp.), avocado(Persea americana), fig (Ficus casica), guava (Psidium guajava), mango(Mangifer indica), olive (Olea europaea), papaya (Carica papaya), cashew(Anacardium occidentale), macadamia (Macadamia intergrifolia) and almond(Prunus amygdalus).

Other preferred plants include C4 grasses such as, in addition to thosementioned above, Andropogon gerardi, Bouteloua curtipendula, B.gracilis, Buchloe dactyloides, Schizachyrium scoparium, Sorghastrumnutans, Sporobolus cryptandrus; C3 grasses such as Elymus canadensis,the legumes Lespedeza capitata and Petalostemum villosum, the forb Asterazureus; and woody plants such as Quercus ellipsoidalis and Q.macrocarpa. Other preferred plants include C3 grasses.

In a preferred embodiment, the plant is an angiosperm.

In an embodiment, the plant is an oilseed plant, preferably an oilseedcrop plant. As used herein, an “oilseed plant” is a plant species usedfor the commercial production of lipid from the seeds of the plant. Theoilseed plant may be, for example, oil-seed rape (such as canola),maize, sunflower, safflower, soybean, sorghum, flax (linseed) or sugarbeet. Furthermore, the oilseed plant may be other Brassicas, cotton,peanut, poppy, rutabaga, mustard, castor bean, sesame, safflower, or nutproducing plants. The plant may produce high levels of lipid in itsfruit such as olive, oil palm or coconut. Horticultural plants to whichthe present invention may be applied are lettuce, endive, or vegetableBrassicas including cabbage, broccoli, or cauliflower. The presentinvention may be applied in tobacco, cucurbits, carrot, strawberry,tomato, or pepper.

In a preferred embodiment, the transgenic plant is homozygous for eachand every gene that has been introduced (transgene) so that its progenydo not segregate for the desired phenotype. The transgenic plant mayalso be heterozygous for the introduced transgene(s), preferablyuniformly heterozygous for the transgene such as for example, in F1progeny which have been gown from hybrid seed. Such plants may provideadvantages such as hybrid vigour, well known in the art.

Where relevant, the transgenic plants may also comprise additionaltransgenes encoding enzymes involved in the production of non-polarlipid such as, but not limited to LPAAT, LPCAT, PAP, or aphospholipid:diacylglycerol acyltransferase (PDAT1, PDAT2 or PDAT3; seefor example, Ghosal et al., 2007), or a combination of two or morethereof. The transgenic plants of the invention may also express oleosinfrom an exogenous polynucleotide.

Transformation of Plants

Transgenic plants can be produced using techniques known in the art,such as those generally described in Slater et al., PlantBiotechnology—The Genetic Manipulation of Plants, Oxford UniversityPress (2003), and Christou and Klee, Handbook of Plant Biotechnology,John Wiley and Sons (2004).

As used herein, the terms “stably transforming”, “stably transformed”and variations thereof refer to the integration of the polynucleotideinto the genome of the cell such that they are transferred to progenycells during cell division without the need for positively selecting fortheir presence. Stable transformants, or progeny thereof, can beselected by any means known in the art such as Southern blots onchromosomal DNA, or in situ hybridization of genomic DNA.

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because DNA can be introduced intocells in whole plant tissues, plant organs, or explants in tissueculture, for either transient expression, or for stable integration ofthe DNA in the plant cell genome. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (see for example, U.S. Pat. No. 5,177,010, U.S. Pat.No. 5,104,310, U.S. Pat. No. 5,004,863, or U.S. Pat. No. 5,159,135). Theregion of DNA to be transferred is defined by the border sequences, andthe intervening DNA (T-DNA) is usually inserted into the plant genome.Further, the integration of the T-DNA is a relatively precise processresulting in few rearrangements. In those plant varieties whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.Preferred Agrobacterium transformation vectors are capable ofreplication in E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., In: Plant DNA InfectiousAgents, Hohn and Schell, eds., Springer-Verlag, New York, pp. 179-203(1985)).

Acceleration methods that may be used include for example,microprojectile bombardment and the like. One example of a method fordelivering transforming nucleic acid molecules to plant cells ismicroprojectile bombardment. This method has been reviewed by Yang etal., Particle Bombardment Technology for Gene Transfer, Oxford Press,Oxford, England (1994). Non-biological particles (microprojectiles) thatmay be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like. A particular advantage ofmicroprojectile bombardment, in addition to it being an effective meansof reproducibly transforming monocots, is that neither the isolation ofprotoplasts, nor the susceptibility of Agrobacterium infection arerequired. An illustrative embodiment of a method for delivering DNA intoZea mays cells by acceleration is a biolistics α-particle deliverysystem, that can be used to propel particles coated with DNA through ascreen such as a stainless steel or Nytex screen, onto a filter surfacecovered with corn cells cultured in suspension. A particle deliverysystem suitable for use with the present invention is the heliumacceleration PDS-1000/He gun available from Bio-Rad Laboratories.

For the bombardment, cells in suspension may be concentrated on filters.Filters containing the cells to be bombarded are positioned at anappropriate distance below the microprojectile stopping plate. Ifdesired, one or more screens are also positioned between the gun and thecells to be bombarded.

Alternatively, immature embryos or other target cells may be arranged onsolid culture medium. The cells to be bombarded are positioned at anappropriate distance below the microprojectile stopping plate. Ifdesired, one or more screens are also positioned between theacceleration device and the cells to be bombarded. Through the use oftechniques set forth herein, one may obtain up to 1000 or more foci ofcells transiently expressing a marker gene. The number of cells in afocus that express the gene product 48 hours post-bombardment oftenrange from one to ten and average one to three.

In bombardment transformation, one may optimize the pre-bombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment are important in this technology. Physicalfactors are those that involve manipulating the DNA/microprojectileprecipitate or those that affect the flight and velocity of either themacro- or microprojectiles. Biological factors include all stepsinvolved in manipulation of cells before and immediately afterbombardment, the osmotic adjustment of target cells to help alleviatethe trauma associated with bombardment, and also the nature of thetransforming DNA such as linearized DNA or intact supercoiled plasmids.It is believed that pre-bombardment manipulations are especiallyimportant for successful transformation of immature embryos.

In another alternative embodiment, plastids can be stably transformed.Methods disclosed for plastid transformation in higher plants includeparticle gun delivery of DNA containing a selectable marker andtargeting of the DNA to the plastid genome through homologousrecombination (U.S. Pat. No. 5,451,513, U.S. Pat. No. 5,545,818, U.S.Pat. No. 5,877,402, U.S. Pat. No. 5,932,479, and WO 99/05265).

Accordingly, it is contemplated that one may wish to adjust variousaspects of the bombardment parameters in small scale studies to fullyoptimize the conditions. One may particularly wish to adjust physicalparameters such as gap distance, flight distance, tissue distance, andhelium pressure. One may also minimize the trauma reduction factors bymodifying conditions that influence the physiological state of therecipient cells and that may therefore influence transformation andintegration efficiencies. For example, the osmotic state, tissuehydration and the subculture stage, or cell cycle of the recipientcells, may be adjusted for optimum transformation. The execution ofother routine adjustments will be known to those of skill in the art inlight of the present disclosure.

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments. Application ofthese systems to different plant varieties depends upon the ability toregenerate that particular plant strain from protoplasts. Illustrativemethods for the regeneration of cereals from protoplasts are described(Fujimura et al., 1985; Toriyama et al., 1986; Abdullah et al., 1986).

Other methods of cell transformation can also be used and include butare not limited to the introduction of DNA into plants by direct DNAtransfer into pollen, by direct injection of DNA into reproductiveorgans of a plant, or by direct injection of DNA into the cells ofimmature embryos followed by the rehydration of desiccated embryos.

The regeneration, development, and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art (Weissbach et al., In: Methods for Plant MolecularBiology, Academic Press, San Diego, Calif., (1988)). This regenerationand growth process typically includes the steps of selection oftransformed cells, culturing those individualized cells through theusual stages of embryonic development through the rooted plantlet stage.Transgenic embryos and seeds are similarly regenerated. The resultingtransgenic rooted shoots are thereafter planted in an appropriate plantgrowth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene is well known in the art. Preferably, the regeneratedplants are self-pollinated to provide homozygous transgenic plants.Otherwise, pollen obtained from the regenerated plants is crossed toseed-grown plants of agronomically important lines. Conversely, pollenfrom plants of these important lines is used to pollinate regeneratedplants. A transgenic plant of the present invention containing a desiredpolynucleotide is cultivated using methods well known to one skilled inthe art.

Methods for transforming dicots, primarily by use of Agrobacteriumtumefaciens, and obtaining transgenic plants have been published forcotton (U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135, U.S. Pat. No.5,518,908), soybean (U.S. Pat. No. 5,569,834, U.S. Pat. No. 5,416,011),Brassica (U.S. Pat. No. 5,463,174), peanut (Cheng et al., 1996), and pea(Grant et al., 1995).

Methods for transformation of cereal plants such as wheat and barley forintroducing genetic variation into the plant by introduction of anexogenous nucleic acid and for regeneration of plants from protoplastsor immature plant embryos are well known in the art, see for example, CA2,092,588, AU 61781/94, AU 667939, U.S. Pat. No. 6,100,447, WO97/048814, U.S. Pat. No. 5,589,617, U.S. Pat. No. 6,541,257, and othermethods are set out in WO 99/14314. Preferably, transgenic wheat orbarley plants are produced by Agrobacterium tumefaciens mediatedtransformation procedures. Vectors carrying the desired polynucleotidemay be introduced into regenerable wheat cells of tissue cultured plantsor explants, or suitable plant systems such as protoplasts.

The regenerable wheat cells are preferably from the scutellum ofimmature embryos, mature embryos, callus derived from these, or themeristematic tissue.

To confirm the presence of the transgenes in transgenic cells andplants, a polymerase chain reaction (PCR) amplification or Southern blotanalysis can be performed using methods known to those skilled in theart. Expression products of the transgenes can be detected in any of avariety of ways, depending upon the nature of the product, and includeWestern blot and enzyme assay. One particularly useful way to quantitateprotein expression and to detect replication in different plant tissuesis to use a reporter gene such as GUS. Once transgenic plants have beenobtained, they may be grown to produce plant tissues or parts having thedesired phenotype. The plant tissue or plant parts, may be harvested,and/or the seed collected. The seed may serve as a source for growingadditional plants with tissues or parts having the desiredcharacteristics. Preferably, the vegetative plant parts are harvested ata time when the yield of non-polar lipids are at their highest. In oneembodiment, the vegetative plant parts are harvested about at the timeof flowering.

A transgenic plant formed using Agrobacterium or other transformationmethods typically contains a single genetic locus on one chromosome.Such transgenic plants can be referred to as being hemizygous for theadded gene(s). More preferred is a transgenic plant that is homozygousfor the added gene(s), that is, a transgenic plant that contains twoadded genes, one gene at the same locus on each chromosome of achromosome pair. A homozygous transgenic plant can be obtained byself-fertilising a hemizygous transgenic plant, germinating some of theseed produced and analyzing the resulting plants for the gene ofinterest.

It is also to be understood that two different transgenic plants thatcontain two independently segregating exogenous genes or loci can alsobe crossed (mated) to produce offspring that contain both sets of genesor loci. Selfing of appropriate F1 progeny can produce plants that arehomozygous for both exogenous genes or loci. Back-crossing to a parentalplant and out-crossing with a non-transgenic plant are alsocontemplated, as is vegetative propagation. Descriptions of otherbreeding methods that are commonly used for different traits and cropscan be found in Fehr, In: Breeding Methods for Cultivar Development,Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987).

Tilling

In one embodiment, TILLING (Targeting Induced Local Lesions IN Genomes)can be used to produce plants in which endogenous genes are knocked out,for example genes encoding a DGAT, sn-1 glycerol-3-phosphateacyltransferase (GPAT), 1-acyl-glycerol-3-phosphate acyltransferase(LPAAT), acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT),phosphatidic acid phosphatase (PAP), or a combination of two or morethereof.

In a first step, introduced mutations such as novel single base pairchanges are induced in a population of plants by treating seeds (orpollen) with a chemical mutagen, and then advancing plants to ageneration where mutations will be stably inherited. DNA is extracted,and seeds are stored from all members of the population to create aresource that can be accessed repeatedly over time.

For a TILLING assay, PCR primers are designed to specifically amplify asingle gene target of interest. Specificity is especially important if atarget is a member of a gene family or part of a polyploid genome. Next,dye-labeled primers can be used to amplify PCR products from pooled DNAof multiple individuals. These PCR products are denatured and reannealedto allow the formation of mismatched base pairs. Mismatches, orheteroduplexes, represent both naturally occurring single nucleotidepolymorphisms (SNPs) (i.e., several plants from the population arelikely to carry the same polymorphism) and induced SNPs (i.e., only rareindividual plants are likely to display the mutation). Afterheteroduplex formation, the use of an endonuclease, such as Cell, thatrecognizes and cleaves mismatched DNA is the key to discovering novelSNPs within a TILLING population.

Using this approach, many thousands of plants can be screened toidentify any individual with a single base change as well as smallinsertions or deletions (1-30 bp) in any gene or specific region of thegenome. Genomic fragments being assayed can range in size anywhere from0.3 to 1.6 kb. At 8-fold pooling, 1.4 kb fragments (discounting the endsof fragments where SNP detection is problematic due to noise) and 96lanes per assay, this combination allows up to a million base pairs ofgenomic DNA to be screened per single assay, making TILLING ahigh-throughput technique. TILLING is further described in Slade andKnauf (2005), and Henikoff et al. (2004).

In addition to allowing efficient detection of mutations,high-throughput TILLING technology is ideal for the detection of naturalpolymorphisms. Therefore, interrogating an unknown homologous DNA byheteroduplexing to a known sequence reveals the number and position ofpolymorphic sites. Both nucleotide changes and small insertions anddeletions are identified, including at least some repeat numberpolymorphisms. This has been called Ecotilling (Comai et al., 2004).

Each SNP is recorded by its approximate position within a fewnucleotides. Thus, each haplotype can be archived based on its mobility.Sequence data can be obtained with a relatively small incremental effortusing aliquots of the same amplified DNA that is used for themismatch-cleavage assay. The left or right sequencing primer for asingle reaction is chosen by its proximity to the polymorphism.Sequencher software performs a multiple alignment and discovers the basechange, which in each case confirmed the gel band.

Ecotilling can be performed more cheaply than full sequencing, themethod currently used for most SNP discovery. Plates containing arrayedecotypic DNA can be screened rather than pools of DNA from mutagenizedplants. Because detection is on gels with nearly base pair resolutionand background patterns are uniform across lanes, bands that are ofidentical size can be matched, thus discovering and genotyping SNPs in asingle step. In this way, ultimate sequencing of the SNP is simple andefficient, made more so by the fact that the aliquots of the same PCRproducts used for screening can be subjected to DNA sequencing.

Enhancing Exogenous RNA Levels and Stabilized Expression

Post-transcriptional gene silencing (PTGS) is a nucleotidesequence-specific defense mechanism that can target both cellular andviral mRNAs for degradation. PTGS occurs in plants or fungi stably ortransiently transformed with a recombinant polynucleotide(s) and resultsin the reduced accumulation of RNA molecules with sequence similarity tothe introduced polynucleotide. “Post-transcriptional” refers to amechanism operating at least partly, but not necessarily exclusively,after production of an initial RNA transcript, for example duringprocessing of the initial RNA transcript, or concomitant with splicingor export of the RNA to the cytoplasm, or within the cytoplasm bycomplexes associated with Argonaute proteins.

RNA molecule levels can be increased, and/or RNA molecule levelsstabilized over numerous generations or under different environmentalconditions, by limiting the expression of a silencing suppressor in astorage organ of a plant or part thereof. As used herein, a “silencingsuppressor” is any polynucleotide or polypeptide that can be expressedin a plant cell that enhances the level of expression product from adifferent transgene in the plant cell, particularly, over repeatedgenerations from the initially transformed plant. In an embodiment, thesilencing suppressor is a viral silencing suppressor or mutant thereof.A large number of viral silencing suppressors are known in the art andinclude, but are not limited to P19, V2, P38, Pe-Po and RPV-P0. Examplesof suitable viral silencing suppressors include those described in WO2010/057246. A silencing suppressor may be stably expressed in a plantor part thereof of the present invention.

As used herein, the term “stably expressed” or variations thereof refersto the level of the RNA molecule being essentially the same or higher inprogeny plants over repeated generations, for example, at least three,at least five, or at least ten generations, when compared tocorresponding plants lacking the exogenous polynucleotide encoding thesilencing suppressor. However, this term(s) does not exclude thepossibility that over repeated generations there is some loss of levelsof the RNA molecule when compared to a previous generation, for example,not less than a 10% loss per generation.

The suppressor can be selected from any source e.g. plant, viral,mammal, etc. The suppressor may be, for example, flock house virus B2,pothos latent virus P14, pothos latent virus AC2, African cassava mosaicvirus AC4, bhendi yellow vein mosaic disease C2, bhendi yellow veinmosaic disease C4, bhendi yellow vein mosaic disease βC1, tomatochlorosis virus p22, tomato chlorosis virus CP, tomato chlorosis virusCPm, tomato golden mosaic virus AL2, tomato leaf curl Java virus βC1,tomato yellow leaf curl virus V2, tomato yellow leaf curl virus-ChinaC2, tomato yellow leaf curl China virus Y10 isolate βC1, tomato yellowleaf curl Israeli isolate V2, mungbean yellow mosaic virus-Vigna AC2,hibiscus chlorotic ringspot virus CP, turnip crinkle virus P38, turnipcrinkle virus CP, cauliflower mosaic virus P6, beet yellows virus p21,citrus tristeza virus p20, citrus tristeza virus p23, citrus tristezavirus CP, cowpea mosaic virus SCP, sweet potato chlorotic stunt virusp22, cucumber mosaic virus 2b, tomato aspermy virus HC-Pro, beet curlytop virus L2, soil borne wheat mosaic virus 19K, barley stripe mosaicvirus Gammab, poa semilatent virus Gammab, peanut clump pecluvirus P15,rice dwarf virus Pns10, curubit aphid borne yellows virus P0, beetwestern yellows virus P0, potato virus X P25, cucumber vein yellowingvirus P1b, plum pox virus HC-Pro, sugarcane mosaic virus HC-Pro, potatovirus Y strain HC-Pro, tobacco etch virus P1/HC-Pro, turnip mosaic virusP1/HC-Pro, cocksfoot mottle virus P1, cocksfoot mottle virus-Norwegianisolate P1, rice yellow mottle virus P1, rice yellow mottlevirus-Nigerian isolate P1, rice hoja blanca virus NS3, rice stripe virusNS3, crucifer infecting tobacco mosaic virus 126K, crucifer infectingtobacco mosaic virus p122, tobacco mosaic virus p122, tobacco mosaicvirus 126, tobacco mosaic virus 130K, tobacco rattle virus 16K, tomatobushy stunt virus P19, tomato spotted wilt virus NSs, apple chloroticleaf spot virus P50, grapevine virus A p10, grapevine leafrollassociated virus-2 homolog of BYV p21, as well as variants/mutantsthereof. The list above provides the virus from which the suppressor canbe obtained and the protein (e.g., B2, P14, etc.), or coding regiondesignation for the suppressor from each particular virus. Othercandidate silencing suppressors may be obtained by examining viralgenome sequences for polypeptides encoded at the same position withinthe viral genome, relative to the structure of a related viral genomecomprising a known silencing suppressor, as is appreciated by a personof skill in the art.

Silencing suppressors can be categorized based on their mode of action.Suppressors such as V2 which preferentially bind to a double-strandedRNA molecule which has overhanging 5′ ends relative to a correspondingdouble-stranded RNA molecule having blunt ends are particularly usefulfor enhancing transgene expression when used in combination with genesilencing (exogenous polynucleotide encoding a dsRNA). Other suppressorssuch as p19 which preferentially bind a dsRNA molecule which is 21 basepairs in length relative to a dsRNA molecule of a different length canalso allow transgene expression in the presence of an exogenouspolynucleotide encoding a dsRNA, but generally to a lesser degree than,for example, V2. This allows the selection of an optimal combination ofdsRNA, silencing suppressor and over-expressed transgene for aparticular purpose. Such optimal combinations can be identified using amethod of the invention.

In an embodiment, the silencing suppressor preferentially binds to adouble-stranded RNA molecule which has overhanging 5′ ends relative to acorresponding double-stranded RNA molecule having blunt ends. In thiscontext, the corresponding double-stranded RNA molecule preferably hasthe same nucleotide sequence as the molecule with the 5′ overhangingends, but without the overhanging 5′ ends. Binding assays are routinelyperformed, for example in in vitro assays, by any method as known to aperson of skill in the art.

Multiple copies of a suppressor may be used. Different suppressors maybe used together (e. g., in tandem).

Essentially any RNA molecule which is desirable to be expressed in aplant storage organ can be co-expressed with the silencing suppressor.The RNA molecule may influence an agronomic trait, insect resistance,disease resistance, herbicide resistance, sterility, graincharacteristics, and the like. The encoded polypeptides may be involvedin metabolism of lipid, starch, carbohydrates, nutrients, etc., or maybe responsible for the synthesis of proteins, peptides, lipids, waxes,starches, sugars, carbohydrates, flavors, odors, toxins, carotenoids.hormones, polymers, flavonoids, storage proteins, phenolic acids,alkaloids, lignins, tannins, celluloses, glycoproteins, glycolipids,etc.

In a particular example, the plants produced increased levels of enzymesfor lipid production in plants such as Brassicas, for example oilseedrape or sunflower, safflower, flax, cotton, soya bean or maize.

Plant Biomass

An increase in the total lipid content of plant biomass equates togreater energy content, making its use in the production of biofuel moreeconomical.

Plant biomass is the organic materials produced by plants, such asleaves, roots, seeds, and stalks. Plant biomass is a complex mixture oforganic materials, such as carbohydrates, fats, and proteins, along withsmall amounts of minerals, such as sodium, phosphorus, calcium, andiron. The main components of plant biomass are carbohydrates(approximately 75%, dry weight) and lignin (approximately 25%), whichcan vary with plant type. The carbohydrates are mainly cellulose orhemicellulose fibers, which impart strength to the plant structure, andlignin, which holds the fibers together. Some plants also store starch(another carbohydrate polymer) and fats as sources of energy, mainly inseeds and roots (such as corn, soybeans, and potatoes).

Plant biomass typically has a low energy density as a result of both itsphysical form and moisture content. This makes it inconvenient andinefficient for storage and transport, and also usually unsuitable foruse without some kind of pre-processing.

There are a range of processes available to convert it into a moreconvenient form including: 1) physical pre-processing (for example,grinding) or 2) conversion by thermal (for example, combustion,gasification, pyrolysis) or chemical (for example, anaerobic digestion,fermentation, composting, transesterification) processes. In this way,the biomass is converted into what can be described as a biomass fuel.

Combustion

Combustion is the process by which flammable materials are allowed toburn in the presence of air or oxygen with the release of heat. Thebasic process is oxidation. Combustion is the simplest method by whichbiomass can be used for energy, and has been used to provide heat. Thisheat can itself be used in a number of ways: 1) space heating, 2) water(or other fluid) heating for central or district heating or processheat, 3) steam raising for electricity generation or motive force. Whenthe flammable fuel material is a form of biomass the oxidation is ofpredominantly the carbon (C) and hydrogen (H) in the cellulose,hemicellulose, lignin, and other molecules present to form carbondioxide (CO₂) and water (H₂O).

Gasification

Gasification is a partial oxidation process whereby a carbon source suchas plant biomass, is broken down into carbon monoxide (CO) and hydrogen(H₂), plus carbon dioxide (CO₂) and possibly hydrocarbon molecules suchas methane (CH₄). If the gasification takes place at a relatively lowtemperature, such as 700° C. to 1000° C., the product gas will have arelatively high level of hydrocarbons compared to high temperaturegasification. As a result it may be used directly, to be burned for heator electricity generation via a steam turbine or, with suitable gasclean up, to run an internal combustion engine for electricitygeneration. The combustion chamber for a simple boiler may be closecoupled with the gasifier, or the producer gas may be cleaned of longerchain hydrocarbons (tars), transported, stored and burned remotely. Agasification system may be closely integrated with a combined cycle gasturbine for electricity generation (IGCC—integrated gasificationcombined cycle). Higher temperature gasification (1200° C. to 1600° C.)leads to few hydrocarbons in the product gas, and a higher proportion ofCO and H₂. This is known as synthesis gas (syngas or biosyngas) as itcan be used to synthesize longer chain hydrocarbons using techniquessuch as Fischer-Tropsch (FT) synthesis. If the ratio of H₂ to CO iscorrect (2:1) FT synthesis can be used to convert syngas into highquality synthetic diesel biofuel which is compatible with conventionalfossil diesel and diesel engines.

Pyrolysis

As used herein, the term “pyrolysis” means a process that uses slowheating in the absence of oxygen to produce gaseous, oil and charproducts from biomass. Pyrolysis is a thermal or thermo-chemicalconversion of lipid-based, particularly triglyceride-based, materials.The products of pyrolysis include gas, liquid and a sold char, with theproportions of each depending upon the parameters of the process. Lowertemperatures (around 400° C.) tend to produce more solid char (slowpyrolysis), whereas somewhat higher temperatures (around 500° C.)produce a much higher proportion of liquid (bio-oil), provided thevapour residence time is kept down to around is or less. After this,secondary reactions take place and increase the gas yield. The bio-oilproduced by fast (higher temperature) pyrolysis is a dark brown, mobileliquid with a heating value about half that of conventional fuel oil. Itcan be burned directly, co-fired, upgraded to other fuels or gasified.

Pyrolysis involves direct thermal cracking of the lipids or acombination of thermal and catalytic cracking. At temperatures of about400-500° C., cracking occurs, producing short chain hydrocarbons such asalkanes, alkenes, alkadienes, aromatics, olefins and carboxylic acid, aswell as carbon monoxide and carbon dioxide.

Four main catalyst types can be used including transition metalcatalysts, molecular sieve type catalysts, activated alumina and sodiumcarbonate (Maher et al., 2007). Examples are given in U.S. Pat. No.4,102,938. Alumina (Al₂O₃) activated by acid is an effective catalyst(U.S. Pat. No. 5,233,109). Molecular sieve catalysts are porous, highlycrystalline structures that exhibit size selectivity, so that moleculesof only certain sizes can pass through. These include zeolite catalystssuch as ZSM-5 or HZSM-5 which are crystalline materials comprising AlO₄and SiO₄ and other silica-alumina catalysts. The activity andselectivity of these catalysts depends on the acidity, pore size andpore shape, and typically operate at 300-500° C. Transition metalcatalysts are described for example in U.S. Pat. No. 4,992,605. Sodiumcarbonate catalyst has been used in the pyrolysis of oils (Dandik andAksoy, 1998).

Transesterification

“Transesterification” as used herein is the conversion of lipids,principally triacylglycerols, into fatty acid methyl esters or ethylesters using short chain alcohols such as methanol or ethanol, in thepresence of a catalyst such as alkali or acid. Methanol is used morecommonly due to low cost and availability. The catalysts may behomogeneous catalysts, heterogeneous catalysts or enzymatic catalysts.Homogeneous catalysts include ferric sulphate followed by KOH.Heterogeneous catalysts include CaO, K₃PO₄, and WO₃/ZrO₂. Enzymaticcatalysts include Novozyme 435 produced from Candida antarctica.

Anaerobic Digestion

Anaerobic digestion is the process whereby bacteria break down organicmaterial in the absence of air, yielding a biogas containing methane.The products of this process are biogas (principally methane (CH₄) andcarbon dioxide (CO₂)), a solid residue (fibre or digestate) that issimilar, but not identical, to compost and a liquid liquor that can beused as a fertilizer. The methane can be burned for heat or electricitygeneration. The solid residue of the anaerobic digestion process can beused as a soil conditioner or alternatively can be burned as a fuel, orgasified.

Anaerobic digestion is typically performed on biological material in anaqueous slurry. However there are an increasing number of dry digesters.Mesophilic digestion takes place between 20° C. and 40° C. and can takea month or two to complete. Thermophilic digestion takes place from50-65° C. and is faster, but the bacteria are more sensitive.

Fermentation

Conventional fermentation processes for the production of bioalcoholmake use of the starch and sugar components of plant crops. Secondgeneration bioalcohol precedes this with acid and/or enzymatichydrolysis of hemicellulose and cellulose into fermentable saccharidesto make use of a much larger proportion of available biomass. Moredetail is provided below under the heading “Fermentation processes forlipid production”.

Composting

Composting is the aerobic decomposition of organic matter bymicroorganisms. It is typically performed on relatively dry materialrather than a slurry. Instead of, or in addition to, collecting theflammable biogas emitted, the exothermic nature of the compostingprocess can be exploited and the heat produced used, usually using aheat pump.

Production of Non-Polar Lipids

Techniques that are routinely practiced in the art can be used toextract, process, purify and analyze the non-polar lipids produced bycells, organisms or parts thereof of the instant invention. Suchtechniques are described and explained throughout the literature insources such as, Fereidoon Shahidi, Current Protocols in Food AnalyticalChemistry, John Wiley & Sons, Inc. (2001) D1.1.1-D1.1.11, and Perez-Vichet al. (1998).

Production of Seedoil

Typically, plant seeds are cooked, pressed, and/or extracted to producecrude seedoil, which is then degummed, refined, bleached, anddeodorized. Generally, techniques for crushing seed are known in theart. For example, oilseeds can be tempered by spraying them with waterto raise the moisture content to, for example, 8.5%, and flaked using asmooth roller with a gap setting of 0.23 to 0.27 mm. Depending on thetype of seed, water may not be added prior to crushing. Application ofheat deactivates enzymes, facilitates further cell rupturing, coalescesthe lipid droplets, and agglomerates protein particles, all of whichfacilitate the extraction process.

In an embodiment, the majority of the seedoil is released by passagethrough a screw press. Cakes expelled from the screw press are thensolvent extracted for example, with hexane, using a heat traced column.Alternatively, crude seedoil produced by the pressing operation can bepassed through a settling tank with a slotted wire drainage top toremove the solids that are expressed with the seedoil during thepressing operation. The clarified seedoil can be passed through a plateand frame filter to remove any remaining fine solid particles. Ifdesired, the seedoil recovered from the extraction process can becombined with the clarified seedoil to produce a blended crude seedoil.

Once the solvent is stripped from the crude seedoil, the pressed andextracted portions are combined and subjected to normal lipid processingprocedures (i.e., degumming, caustic refining, bleaching, anddeodorization).

In an embodiment, the oil and/or protein content of the seed is analysedby near-infrared reflectance spectroscopy as described in Hom et al.(2007).

Degumming

Degumming is an early step in the refining of oils and its primarypurpose is the removal of most of the phospholipids from the oil, whichmay be present as approximately 1-2% of the total extracted lipid.Addition of ˜2% of water, typically containing phosphoric acid, at70-80° C. to the crude oil results in the separation of most of thephospholipids accompanied by trace metals and pigments. The insolublematerial that is removed is mainly a mixture of phospholipids andtriacylglycerols and is also known as lecithin. Degumming can beperformed by addition of concentrated phosphoric acid to the crudeseedoil to convert non-hydratable phosphatides to a hydratable form, andto chelate minor metals that are present. Gum is separated from theseedoil by centrifugation. The seedoil can be refined by addition of asufficient amount of a sodium hydroxide solution to titrate all of thefatty acids and removing the soaps thus formed.

Alkali Refining

Alkali refining is one of the refining processes for treating crude oil,sometimes also referred to as neutralization. It usually followsdegumming and precedes bleaching. Following degumming, the seedoil cantreated by the addition of a sufficient amount of an alkali solution totitrate all of the fatty acids and phosphoric acids, and removing thesoaps thus formed. Suitable alkaline materials include sodium hydroxide,potassium hydroxide, sodium carbonate, lithium hydroxide, calciumhydroxide, calcium carbonate and ammonium hydroxide. This process istypically carried out at room temperature and removes the free fattyacid fraction. Soap is removed by centrifugation or by extraction into asolvent for the soap, and the neutralised oil is washed with water. Ifrequired, any excess alkali in the oil may be neutralized with asuitable acid such as hydrochloric acid or sulphuric acid.

Bleaching

Bleaching is a refining process in which oils are heated at 90-120° C.for 10-30 minutes in the presence of a bleaching earth (0.2-2.0%) and inthe absence of oxygen by operating with nitrogen or steam or in avacuum. This step in oil processing is designed to remove unwantedpigments (carotenoids, chlorophyll, gossypol etc), and the process alsoremoves oxidation products, trace metals, sulphur compounds and tracesof soap.

Deodorization

Deodorization is a treatment of oils and fats at a high temperature(200-260° C.) and low pressure (0.1-1 mm Hg). This is typically achievedby introducing steam into the seedoil at a rate of about 0.1ml/minute/100 ml of seedoil. Deodorization can be performed by heatingthe seedoil to 260° C. under vacuum, and slowly introducing steam intothe seedoil at a rate of about 0.1 ml/minute/100 ml of seedoil. Afterabout 30 minutes of sparging, the seedoil is allowed to cool undervacuum. The seedoil is typically transferred to a glass container andflushed with argon before being stored under refrigeration. If theamount of seedoil is limited, the seedoil can be placed under vacuum forexample, in a Parr reactor and heated to 260° C. for the same length oftime that it would have been deodorized. This treatment improves thecolour of the seedoil and removes a majority of the volatile substancesor odorous compounds including any remaining free fatty acids,monoacylglycerols and oxidation products.

Winterisation

Winterization is a process sometimes used in commercial production ofoils for the separation of oils and fats into solid (stearin) and liquid(olein) fractions by crystallization at sub-ambient temperatures. It wasapplied originally to cottonseed oil to produce a solid-free product. Itis typically used to decrease the saturated fatty acid content of oils.

Plant Biomass for the Production of Lipids

Parts of plants involved in photosynthesis (e.g., and stems and leavesof higher plants and aquatic plants such as algae) can also be used toproduce lipid. Independent of the type of plant, there are severalmethods for extracting lipids from green biomass. One way is physicalextraction, which often does not use solvent extraction. It is a“traditional” way using several different types of mechanicalextraction. Expeller pressed extraction is a common type, as are thescrew press and ram press extraction methods. The amount of lipidextracted using these methods varies widely, depending upon the plantmaterial and the mechanical process employed. Mechanical extraction istypically less efficient than solvent extraction described below.

In solvent extraction, an organic solvent (e.g., hexane) is mixed withat least the genetically modified plant green biomass, preferably afterthe green biomass is dried and ground. Of course, other parts of theplant besides the green biomass (e.g., lipid-containing seeds) can beground and mixed in as well. The solvent dissolves the lipid in thebiomass and the like, which solution is then separated from the biomassby mechanical action (e.g., with the pressing processes above). Thisseparation step can also be performed by filtration (e.g., with a filterpress or similar device) or centrifugation etc. The organic solvent canthen be separated from the non-polar lipid (e.g., by distillation). Thissecond separation step yields non-polar lipid from the plant and canyield a re-usable solvent if one employs conventional vapor recovery.

If, for instance, vegetative tissue as described herein, is not to beused immediately to extract, and/or process, the lipid it is preferablyhandled post-harvest to ensure the lipid content does not decrease, orsuch that any decrease in lipid content is minimized as much as possible(see, for example, Christie, 1993). In one embodiment, the vegetativetissue is frozen as soon as possible after harvesting using, forexample, dry ice or liquid nitrogen. In another embodiment, thevegetative tissue is stored at a cold temperature, for example −20° C.or −60° C. in an atmosphere of nitrogen.

Algae for the Production of Lipids

Algae can produce 10 to 100 times as much mass as terrestrial plants ina year. In addition to being a prolific organism, algae are also capableof producing oils and starches that can be converted into biofuels.

The specific algae most useful for biofuel production are known asmicroalgae, consisting of small, often unicellular, types. These algaecan grow almost anywhere. With more than 100,000 known species ofdiatoms (a type of alga), 40,000 known species of green plant-likealgae, and smaller numbers of other algae species, algae will growrapidly in nearly any environment, with almost any kind of water.Specifically, useful algae can be grown in marginal areas with limitedor poor quality water, such as in the arid and mostly empty regions ofthe American Southwest. These areas also have abundant sunshine forphotosynthesis. In short, algae can be an ideal organism for productionof biofuels—efficient growth, needing no premium land or water, notcompeting with food crops, needing much smaller amounts of land thanfood crops, and storing energy in a desirable form.

Algae can store energy in its cell structure in the form of either oilor starch. Stored oil can be as much as 60% of the weight of the algae.Certain species which are highly prolific in oil or starch productionhave been identified, and growing conditions have been tested. Processesfor extracting and converting these materials to fuels have also beendeveloped.

The most common oil-producing algae can generally include, or consistessentially of, the diatoms (bacillariophytes), green algae(chlorophytes), blue-green algae (cyanophytes), and golden-brown algae(chrysophytes). In addition a fifth group known as haptophytes may beused. Groups include brown algae and heterokonts. Specific non-limitingexamples algae include the Classes: Chlorophyceae, Eustigmatophyceae,Prymnesiophyceae, Bacillariophyceae. Bacillariophytes capable of oilproduction include the genera Amphipleura, Amphora, Chaetoceros,Cyclotella, Cymbella, Fragilaria, Hantzschia, Navicula, Nitzschia,Phaeodactylum, and Thalassiosira. Specific non-limiting examples ofchlorophytes capable of oil production include Ankistrodesmus,Botryococcus, Chlorella, Chlorococcum, Dunaliella, Monoraphidium,Oocystis, Scenedesmus, and Tetraselmis In one aspect, the chlorophytescan be Chlorella or Dunaliella. Specific non-limiting examples ofcyanophytes capable of oil production include Oscillatoria andSynechococcus. A specific example of chrysophytes capable of oilproduction includes Boekelovia. Specific non-limiting examples ofhaptophytes include Isochrysis and Pleurochrysis.

Specific algae useful in the present invention include, for example,Chlamydomonas sp. such as Chlamydomonas reinhardtii, Dunaliella sp. suchas Dunaliella sauna, Dunaliella tertiolecta, D. acidophila, D. bardawil,D. bioculata, D. lateralis, D. maritima, D. minuta, D. parva, D.peircei, D. polymorpha, D. primolecta, D. pseudosalina, D. quartolecta.D. viridis, Haematococcus sp., Chlorella sp. such as Chlorella vulgaris,Chlorella sorokiniana or Chlorella protothecoides, Thraustochytrium sp.,Schizochytrium sp., Volvox sp, Nannochloropsis sp., Botryococcus brauniiwhich can contain over 60 wt % lipid, Phaeodactylum tricornutum,Thalassiosira pseudonana, Isochrysis sp., Pavlova sp., Chlorococcum sp,Ellipsoidion sp., Neochloris sp., Scenedesmus sp.

Further, the oil-producing algae of the present invention can include acombination of an effective amount of two or more strains in order tomaximize benefits from each strain. As a practical matter, it can bedifficult to achieve 100% purity of a single strain of algae or acombination of desired algae strains. However, when discussed herein,the oil-producing algae is intended to cover intentionally introducedstrains of algae, while foreign strains are preferably minimized andkept below an amount which would detrimentally affect yields of desiredoil-producing algae and algal oil. Undesirable algae strains can becontrolled and/or eliminated using any number of techniques. Forexample, careful control of the growth environment can reduceintroduction of foreign strains. Alternatively, or in addition to othertechniques, a virus selectively chosen to specifically target only theforeign strains can be introduced into the growth reservoirs in anamount which is effective to reduce and/or eliminate the foreign strain.An appropriate virus can be readily identified using conventionaltechniques. For example, a sample of the foreign algae will most ofteninclude small amounts of a virus which targets the foreign algae. Thisvirus can be isolated and grown in order to produce amounts which wouldeffectively control or eliminate the foreign algae population among themore desirable oil-producing algae.

Algaculture is a form of aquaculture involving the farming of species ofalgae (including microalgae, also referred to as phytoplankton,microphytes, or planktonic algae, and macroalgae, commonly known asseaweed).

Commercial and industrial algae cultivation has numerous uses, includingproduction of food ingredients, food, and algal fuel.

Mono or mixed algal cultures can be cultured in open-ponds (such asraceway-type ponds and lakes) or photobioreactors.

Algae can be harvested using microscreens, by centrifugation, byflocculation (using for example, chitosan, alum and ferric chloride) andby froth flotation. Interrupting the carbon dioxide supply can causealgae to flocculate on its own, which is called “autoflocculation”. Infroth flotation, the cultivator aerates the water into a froth, and thenskims the algae from the top. Ultrasound and other harvesting methodsare currently under development.

Lipid may be separated from the algae by mechanical crushing. When algaeis dried it retains its lipid content, which can then be “pressed” outwith an oil press. Since different strains of algae vary widely in theirphysical attributes, various press configurations (screw, expeller,piston, etc.) work better for specific algae types.

Osmotic shock is sometimes used to release cellular components such aslipid from algae. Osmotic shock is a sudden reduction in osmoticpressure and can cause cells in a solution to rupture.

Ultrasonic extraction can accelerate extraction processes, in particularenzymatic extraction processes employed to extract lipid from algae.Ultrasonic waves are used to create cavitation bubbles in a solventmaterial. When these bubbles collapse near the cell walls, the resultingshock waves and liquid jets cause those cells walls to break and releasetheir contents into a solvent.

Chemical solvents (for example, hexane, benzene, petroleum ether) areoften used in the extraction of lipids from algae. Soxhlet extractioncan be use to extract lipids from algae through repeated washing, orpercolation, with an organic solvent under reflux in a specialglassware.

Enzymatic extraction may be used to extract lipids from algae. Ezymaticextraction uses enzymes to degrade the cell walls with water acting asthe solvent. The enzymatic extraction can be supported byultrasonication.

Supercritical CO₂ can also be used as a solvent. In this method, CO₂ isliquefied under pressure and heated to the point that it becomessupercritical (having properties of both a liquid and a gas), allowingit to act as a solvent.

Fermentation Processes for Lipid Production

As used herein, the term the “fermentation process” refers to anyfermentation process or any process comprising a fermentation step. Afermentation process includes, without limitation, fermentationprocesses used to produce alcohols (e.g., ethanol, methanol, butanol),organic acids (e.g., citric acid, acetic acid, itaconic acid, lacticacid, gluconic acid), ketones (e.g., acetone), amino acids (e.g.,glutamic acid), gases (e.g., H₂ and CO₂), antibiotics (e.g., penicillinand tetracycline), enzymes, vitamins (e.g., riboflavin, beta-carotene),and hormones. Fermentation processes also include fermentation processesused in the consumable alcohol industry (e.g., beer and wine), dairyindustry (e.g., fermented dairy products), leather industry and tobaccoindustry. Preferred fermentation processes include alcohol fermentationprocesses, as are well known in the art. Preferred fermentationprocesses are anaerobic fermentation processes, as are well known in theart. Suitable fermenting cells, typically microorganisms that are ableto ferment, that is, convert, sugars such as glucose or maltose,directly or indirectly into the desired fermentation product.

Examples of fermenting microorganisms include fungal organisms such asyeast, preferably an oleaginous organism. As used herein, an “oleaginousorganism” is one which accumulates at least 25% of its dry weight astriglycerides. As used herein, “yeast” includes Saccharomyces spp.,Saccharomyces cerevisiae, Saccharomyces carlbergensis, Candida spp.,Kluveromyces spp., Pichia spp., Hansenula spp., Trichoderma spp.,Lipomyces starkey, and Yarrowia lipolytica. Preferred yeast includeYarrowia lipolytica or other oleaginous yeasts and strains of theSaccharomyces spp., and in particular, Saccharomyces cerevisiae.

In one embodiment, the fermenting microorganism is a transgenic organismthat comprises one or more exogenous polynucleotides, wherein thetransgenic organism has an increased level of one or more non-polarlipids when compared to a corresponding organism lacking the one or moreexogenous polynucleotides. The transgenic microorganism is preferablygrown under conditions that optimize activity of fatty acid biosyntheticgenes and fatty acid acyltransferase genes. This leads to production ofthe greatest and the most economical yield of lipid. In general, mediaconditions that may be optimized include the type and amount of carbonsource, the type and amount of nitrogen source, the carbon-to-nitrogenratio, the oxygen level, growth temperature, pH, length of the biomassproduction phase, length of the lipid accumulation phase and the time ofcell harvest.

Fermentation media must contain a suitable carbon source. Suitablecarbon sources may include, but are not limited to: monosaccharides(e.g., glucose, fructose), disaccharides (e.g., lactose, sucrose),oligosaccharides, polysaccharides (e.g., starch, cellulose or mixturesthereof), sugar alcohols (e.g., glycerol) or mixtures from renewablefeedstocks (e.g., cheese whey permeate, cornsteep liquor, sugar beetmolasses, barley malt). Additionally, carbon sources may includealkanes, fatty acids, esters of fatty acids, monoglycerides,diglycerides, triglycerides, phospholipids and various commercialsources of fatty acids including vegetable oils (e.g., soybean oil) andanimal fats. Additionally, the carbon substrate may include one-carbonsubstrates (e.g., carbon dioxide, methanol, formaldehyde, formate,carbon-containing amines) for which metabolic conversion into keybiochemical intermediates has been demonstrated. Hence it iscontemplated that the source of carbon utilized in the present inventionmay encompass a wide variety of carbon-containing substrates and willonly be limited by the choice of the host microorganism. Although all ofthe above mentioned carbon substrates and mixtures thereof are expectedto be suitable in the present invention, preferred carbon substrates aresugars and/or fatty acids. Most preferred is glucose and/or fatty acidscontaining between 10-22 carbons. Nitrogen may be supplied from aninorganic (e.g., (NH₄)₂SO₄) or organic source (e.g., urea, glutamate).In addition to appropriate carbon and nitrogen sources, the fermentationmedia may also contain suitable minerals, salts, cofactors, buffers,vitamins and other components known to those skilled in the art suitablefor the growth of the microorganism and promotion of the enzymaticpathways necessary for lipid production.

A suitable pH range for the fermentation is typically between about pH4.0 to pH 8.0, wherein pH 5.5 to pH 7.0 is preferred as the range forthe initial growth conditions. The fermentation may be conducted underaerobic or anaerobic conditions, wherein microaerobic conditions arepreferred.

Typically, accumulation of high levels of lipid in the cells ofoleaginous microorganisms requires a two-stage process, since themetabolic state must be “balanced” between growth and synthesis/storageof fats. Thus, most preferably, a two-stage fermentation process isnecessary for the production of lipids in microorganisms. In thisapproach, the first stage of the fermentation is dedicated to thegeneration and accumulation of cell mass and is characterized by rapidcell growth and cell division. In the second stage of the fermentation,it is preferable to establish conditions of nitrogen deprivation in theculture to promote high levels of lipid accumulation. The effect of thisnitrogen deprivation is to reduce the effective concentration of AMP inthe cells, thereby reducing the activity of the NAD-dependent isocitratedehydrogenase of mitochondria. When this occurs, citric acid willaccumulate, thus forming abundant pools of acetyl-CoA in the cytoplasmand priming fatty acid synthesis. Thus, this phase is characterized bythe cessation of cell division followed by the synthesis of fatty acidsand accumulation of TAGs.

Although cells are typically grown at about 30° C., some studies haveshown increased synthesis of unsaturated fatty acids at lowertemperatures. Based on process economics, this temperature shift shouldlikely occur after the first phase of the two-stage fermentation, whenthe bulk of the microorganism's growth has occurred.

It is contemplated that a variety of fermentation process designs may beapplied, where commercial production of lipids using the instant nucleicacids is desired. For example, commercial production of lipid from arecombinant microbial host may be produced by a batch, fed-batch orcontinuous fermentation process.

A batch fermentation process is a closed system wherein the mediacomposition is set at the beginning of the process and not subject tofurther additions beyond those required for maintenance of pH and oxygenlevel during the process. Thus, at the beginning of the culturingprocess the media is inoculated with the desired organism and growth ormetabolic activity is permitted to occur without adding additionalsubstrates (i.e., carbon and nitrogen sources) to the medium. In batchprocesses the metabolite and biomass compositions of the system changeconstantly up to the time the culture is terminated. In a typical batchprocess, cells moderate through a static lag phase to a high-growth logphase and finally to a stationary phase, wherein the growth rate isdiminished or halted. Left untreated, cells in the stationary phase willeventually die. A variation of the standard batch process is thefed-batch process, wherein the substrate is continually added to thefermentor over the course of the fermentation process. A fed-batchprocess is also suitable in the present invention. Fed-batch processesare useful when catabolite repression is apt to inhibit the metabolismof the cells or where it is desirable to have limited amounts ofsubstrate in the media at any one time. Measurement of the substrateconcentration in fed-batch systems is difficult and therefore may beestimated on the basis of the changes of measurable factors such as pH,dissolved oxygen and the partial pressure of waste gases (e.g., CO₂).Batch and fed-batch culturing methods are common and well known in theart and examples may be found in Brock, In Biotechnology: A Textbook ofIndustrial Microbiology, 2.sup.nd ed., Sinauer Associates, Sunderland,Mass., (1989); or Deshpande (1992).

Commercial production of lipid using the instant cells may also beaccomplished by a continuous fermentation process, wherein a definedmedia is continuously added to a bioreactor while an equal amount ofculture volume is removed simultaneously for product recovery.Continuous cultures generally maintain the cells in the log phase ofgrowth at a constant cell density. Continuous or semi-continuous culturemethods permit the modulation of one factor or any number of factorsthat affect cell growth or end product concentration. For example, oneapproach may limit the carbon source and allow all other parameters tomoderate metabolism. In other systems, a number of factors affectinggrowth may be altered continuously while the cell concentration,measured by media turbidity, is kept constant. Continuous systems striveto maintain steady state growth and thus the cell growth rate must bebalanced against cell loss due to media being drawn off the culture.Methods of modulating nutrients and growth factors for continuousculture processes, as well as techniques for maximizing the rate ofproduct formation, are well known in the art of industrial microbiologyand a variety of methods are detailed by Brock, supra.

Fatty acids, including PUFAs, may be found in the host microorganism asfree fatty acids or in esterified forms such as acylglycerols,phospholipids, sulfolipids or glycolipids, and may be extracted from thehost cell through a variety of means well-known in the art.

In general, means for the purification of fatty acids, including PUFAs,may include extraction with organic solvents, sonication, supercriticalfluid extraction (e.g., using carbon dioxide), saponification andphysical means such as presses, or combinations thereof. Of particularinterest is extraction with methanol and chloroform in the presence ofwater (Bligh and Dyer, 1959). Where desirable, the aqueous layer can beacidified to protonate negatively-charged moieties and thereby increasepartitioning of desired products into the organic layer. Afterextraction, the organic solvents can be removed by evaporation under astream of nitrogen. When isolated in conjugated forms, the products maybe enzymatically or chemically cleaved to release the free fatty acid ora less complex conjugate of interest, and can then be subject to furthermanipulations to produce a desired end product. Desirably, conjugatedforms of fatty acids are cleaved with potassium hydroxide.

If further purification is necessary, standard methods can be employed.Such methods may include extraction, treatment with urea, fractionalcrystallization, HPLC, fractional distillation, silica gelchromatography, high-speed centrifugation or distillation, orcombinations of these techniques. Protection of reactive groups such asthe acid or alkenyl groups, may be done at any step through knowntechniques (e.g., alkylation, iodination). Methods used includemethylation of the fatty acids to produce methyl esters. Similarly,protecting groups may be removed at any step. Desirably, purification offractions containing GLA, STA, ARA, DHA and EPA may be accomplished bytreatment with urea and/or fractional distillation.

An example of the use of plant biomass for the production of a biomassslurry using yeast is described in WO 2011/100272.

Uses of Lipids

The lipids produced by the methods described have a variety of uses. Insome embodiments, the lipids are used as food oils. In otherembodiments, the lipids are refined and used as lubricants or for otherindustrial uses such as the synthesis of plastics. In some preferredembodiments, the lipids are refined to produce biodiesel.

Biofuel

As used herein the term “biofuel” includes biodiesel and bioalcohol.Biodiesel can be made from oils derived from plants, algae and fungi.Bioalcohol is produced from the fermentation of sugar. This sugar can beextracted directly from plants (e.g., sugarcane), derived from plantstarch (e.g., maize or wheat) or made from cellulose (e.g., wood, leavesor stems).

Biofuels currently cost more to produce than petroleum fuels. Inaddition to processing costs, biofuel crops require planting,fertilising, pesticide and herbicide applications, harvesting andtransportation. Plants, algae and fungi of the present invention mayreduce production costs of biofuel.

General methods for the production of biofuel can be found in, forexample, Maher and Bressler, 2007; Greenwell et al., 2010; Karmakar etal., 2010; Alonso et al., 2010; Lee and Mohamed, 2010; Liu et al.,2010a; Gong and Jiang, 2011; Endalew et al., 2011; Semwal et al., 2011.

Bioalcohol

The production of biologically produced alcohols, for example, ethanol,propanol and butanol is well known. Ethanol is the most commonbioalcohol.

The basic steps for large scale production of ethanol are: 1) microbial(for example, yeast) fermentation of sugars, 2) distillation, 3)dehydration, and optionally 4) denaturing. Prior to fermentation, somecrops require saccharification or hydrolysis of carbohydrates such ascellulose and starch into sugars. Saccharification of cellulose iscalled cellulolysis. Enzymes can be used to convert starch into sugar.

Fermentation

Bioalcohol is produced by microbial fermentation of the sugar. Microbialfermentation will currently only work directly with sugars. Two majorcomponents of plants, starch and cellulose, are both made up of sugars,and can in principle be converted to sugars for fermentation.

Distillation

For the ethanol to be usable as a fuel, the majority of the water mustbe removed. Most of the water is removed by distillation, but the purityis limited to 95-96% due to the formation of a low-boiling water-ethanolazeotrope with maximum (95.6% m/m (96.5% v/v) ethanol and 4.4% m/m (3.5%v/v) water). This mixture is called hydrous ethanol and can be used as afuel alone, but unlike anhydrous ethanol, hydrous ethanol is notmiscible in all ratios with gasoline, so the water fraction is typicallyremoved in further treatment in order to burn in combination withgasoline in gasoline engines.

Dehydration

Water can be removed from an azeotropic ethanol/water mixture bydehydration. Azeotropic distillation, used in many early fuel ethanolplants, consists of adding benzene or cyclohexane to the mixture. Whenthese components are added to the mixture, it forms a heterogeneousazeotropic mixture in vapor-liquid-liquid equilibrium, which whendistilled produces anhydrous ethanol in the column bottom, and a vapormixture of water and cyclohexane/benzene. When condensed, this becomes atwo-phase liquid mixture. Another early method, called extractivedistillation, consists of adding a ternary component which will increaseethanol's relative volatility. When the ternary mixture is distilled, itwill produce anhydrous ethanol on the top stream of the column.

A third method has emerged and has been adopted by the majority ofmodern ethanol plants. This new process uses molecular sieves to removewater from fuel ethanol. In this process, ethanol vapor under pressurepasses through a bed of molecular sieve beads. The bead's pores aresized to allow absorption of water while excluding ethanol. After aperiod of time, the bed is regenerated under vacuum or in the flow ofinert atmosphere (e.g. N₂) to remove the absorbed water. Two beds areoften used so that one is available to absorb water while the other isbeing regenerated.

Biodiesel

The production of biodiesel, or alkyl esters, is well known. There arethree basic routes to ester production from lipids: 1) Base catalysedtransesterification of the lipid with alcohol; 2) Direct acid catalysedesterification of the lipid with methanol; and 3) Conversion of thelipid to fatty acids, and then to alkyl esters with acid catalysis.

Any method for preparing fatty acid alkyl esters and glyceryl ethers (inwhich one, two or three of the hydroxy groups on glycerol areetherified) can be used. For example, fatty acids can be prepared, forexample, by hydrolyzing or saponifying triglycerides with acid or basecatalysts, respectively, or using an enzyme such as a lipase or anesterase. Fatty acid alkyl esters can be prepared by reacting a fattyacid with an alcohol in the presence of an acid catalyst. Fatty acidalkyl esters can also be prepared by reacting a triglyceride with analcohol in the presence of an acid or base catalyst. Glycerol ethers canbe prepared, for example, by reacting glycerol with an alkyl halide inthe presence of base, or with an olefin or alcohol in the presence of anacid catalyst.

In some preferred embodiments, the lipids are transesterified to producemethyl esters and glycerol. In some preferred embodiments, the lipidsare reacted with an alcohol (such as methanol or ethanol) in thepresence of a catalyst (for example, potassium or sodium hydroxide) toproduce alkyl esters. The alkyl esters can be used for biodiesel orblended with petroleum based fuels.

The alkyl esters can be directly blended with diesel fuel, or washedwith water or other aqueous solutions to remove various impurities,including the catalysts, before blending. It is possible to neutralizeacid catalysts with base. However, this process produces salt. To avoidengine corrosion, it is preferable to minimize the salt concentration inthe fuel additive composition. Salts can be substantially removed fromthe composition, for example, by washing the composition with water.

In another embodiment, the composition is dried after it is washed, forexample, by passing the composition through a drying agent such ascalcium sulfate.

In yet another embodiment, a neutral fuel additive is obtained withoutproducing salts or using a washing step, by using a polymeric acid, suchas Dowex 50™, which is a resin that contains sulfonic acid groups. Thecatalyst is easily removed by filtration after the esterification andetherification reactions are complete.

Plant Triacylglycerols as a Biofuel Source

Use of plant triacylglycerols for the production of biofuel is reviewedin Durrett et al. (2008). Briefly, plant oils are primarily composed ofvarious triacylglycerols (TAGs), molecules that consist of three fattyacid chains (usually 18 or 16 carbons long) esterified to glycerol. Thefatty acyl chains are chemically similar to the aliphatic hydrocarbonsthat make up the bulk of the molecules found in petrol and diesel. Thehydrocarbons in petrol contain between 5 and 12 carbon atoms permolecule, and this volatile fuel is mixed with air and ignited with aspark in a conventional engine. In contrast, diesel fuel componentstypically have 10-15 carbon atoms per molecule and are ignited by thevery high compression obtained in a diesel engine. However, most plantTAGs have a viscosity range that is much higher than that ofconventional diesel: 17.3-32.9 mm²s⁻¹ compared to 1.9-4.1 mm^(2s-1),respectively (ASTM D975; Knothe and Steidley, 2005). This higherviscosity results in poor fuel atomization in modern diesel engines,leading to problems derived from incomplete combustion such as carbondeposition and coking (Ryan et al., 1984). To overcome this problem,TAGs are converted to less viscous fatty acid esters by esterificationwith a primary alcohol, most commonly methanol. The resulting fuel iscommonly referred to as biodiesel and has a dynamic viscosity range from1.9 to 6.0 mm²S⁻¹ (ASTM D6751). The fatty acid methyl esters (FAMEs)found in biodiesel have a high energy density as reflected by their highheat of combustion, which is similar, if not greater, than that ofconventional diesel (Knothe, 2005). Similarly, the cetane number (ameasure of diesel ignition quality) of the FAMEs found in biodieselexceeds that of conventional diesel (Knothe, 2005).

Plant oils are mostly composed of five common fatty acids, namelypalmitate (16:0), stearate (18:0), oleate (18:1), linoleate (18:2) andlinolenate (18:3), although, depending on the particular species, longeror shorter fatty acids may also be major constituents. These fatty acidsdiffer from each other in terms of acyl chain length and number ofdouble bonds, leading to different physical properties. Consequently,the fuel properties of biodiesel derived from a mixture of fatty acidsare dependent on that composition. Altering the fatty acid profile cantherefore improve fuel properties of biodiesel such as cold-temperatureflow characteristics, oxidative stability and NOx emissions. Alteringthe fatty acid composition of TAGs may reduce the viscosity of the plantoils, eliminating the need for chemical modification, thus improving thecost-effectiveness of biofuels.

Most plant oils are derived from triacylglycerols stored in seeds.However, the present invention provides methods for also increasing oilcontent in vegetative tissues. The plant tissues of the presentinvention have an increased total lipid yield. Furthermore, the level ofoleic acid is increased significantly while the polyunsaturated fattyacid alpha linolenic acid was reduced.

Once a leaf is developed, it undergoes a developmental change from sink(absorbing nutrients) to source (providing sugars). In food crops, mostsugars are translocated out of source leaves to support growth of newleaves, roots and fruits. Because translocation of carbohydrate is anactive process, there is a loss of carbon and energy duringtranslocation. Furthermore, after the developing seed takes up carbonfrom the plant, there are additional carbon and energy losses associatedwith the conversion of carbohydrate into the oil, protein or other majorcomponents of the seed (Goffman et al., 2005). Plants of the presentinvention increase the energy content of leaves and/or stems such thatthe whole above-ground plant may be harvested and used to producebiofuel.

Algae as a Biofuel Source

Algae store oil inside the cell body, sometimes but not always invesicles. This oil can be recovered in several relatively simple ways,including solvents, heat, and/or pressure. However, these methodstypically recover only about 80% to 90% of the stored oil. Processeswhich offer more effective oil extraction methods which can recoverclose to 100% of the stored oil at low cost as known in the art. Theseprocesses include or consist of depolymerizing, such as biologicallybreaking the walls of the algal cell and/or oil vesicles, if present, torelease the oil from the oil-producing algae.

In addition, a large number of viruses exist which invade and rupturealgae cells, and can thereby release the contents of the cell inparticular stored oil or starch. Such viruses are an integral part ofthe algal ecosystem, and many of the viruses are specific to a singletype of algae. Specific examples of such viruses include the chlorellavirus PBCV-1 (Paramecium Bursaria Chlorella Virus) which is specific tocertain Chlorella algae, and cyanophages such as SM-1, P-60, and AS-1specific to the blue-green algae Synechococcus. The particular virusselected will depend on the particular species of algae to be used inthe growth process. One aspect of the present invention is the use ofsuch a virus to rupture the algae so that oil contained inside the algaecell wall can be recovered. In another detailed aspect of the presentinvention, a mixture of biological agents can be used to rupture thealgal cell wall and/or oil vesicles.

Mechanical crushing, for example, an expeller or press, a hexane orbutane solvent recovery step, supercritical fluid extraction, or thelike can also be useful in extracting the oil from oil vesicles of theoil-producing algae. Alternatively, mechanical approaches can be used incombination with biological agents in order to improve reaction ratesand/or separation of materials. Regardless of the particular biologicalagent or agents chosen such can be introduced in amounts which aresufficient to serve as the primary mechanism by which algal oil isreleased from oil vesicles in the oil-producing algae, i.e. not a merelyincidental presence of any of these.

Once the oil has been released from the algae it can be recovered orseparated 16 from a slurry of algae debris material, for example,cellular residue, oil, enzyme, by-products, etc. This can be done bysedimentation or centrifugation, with centrifugation generally beingfaster. Starch production can follow similar separation processes.

An algal feed can be formed from a biomass feed source as well as analgal feed source. Biomass from either algal or terrestrial sources canbe depolymerized in a variety of ways such as, but not limited tosaccharification, hydrolysis or the like. The source material can bealmost any sufficiently voluminous cellulose, lignocellulose,polysaccharide or carbohydrate, glycoprotein, or other material makingup the cell wall of the source material.

The fermentation stage can be conventional in its use of yeast toferment sugar to alcohol. The fermentation process produces carbondioxide, alcohol, and algal husks. All of these products can be usedelsewhere in the process and systems of the present invention, withsubstantially no unused material or wasted heat. Alternatively, ifethanol is so produced, it can be sold as a product or used to produceethyl acetate for the transesterification process. Similarconsiderations would apply to alcohols other than ethanol.

Algal oil can be converted to biodiesel through a process of directhydrogenation or transesterification of the algal oil. Algal oil is in asimilar form as most vegetable oils, which are in the form oftriglycerides. A triglyceride consists of three fatty acid chains, oneattached to each of the three carbon atoms in a glycerol backbone. Thisform of oil can be burned directly. However, the properties of the oilin this form are not ideal for use in a diesel engine, and withoutmodification, the engine will soon run poorly or fail. In accordancewith the present invention, the triglyceride is converted intobiodiesel, which is similar to but superior to petroleum diesel fuel inmany respects.

One process for converting the triglyceride to biodiesel istransesterification, and includes reacting the triglyceride with alcoholor other acyl acceptor to produce free fatty acid esters and glycerol.The free fatty acids are in the form of fatty acid alkyl esters (FAAE).

With the chemical process, additional steps are needed to separate thecatalyst and clean the fatty acids. In addition, if ethanol is used asthe acyl acceptor, it must be essentially dry to prevent production ofsoap via saponification in the process, and the glycerol must bepurified. The biological process, by comparison, can accept ethanol in aless dry state and the cleaning and purification of the biodiesel andglycerol are much easier.

Transesterification often uses a simple alcohol, typically methanolderived from petroleum. When methanol is used the resultant biodiesel iscalled fatty acid methyl ester (FAME) and most biodiesel sold today,especially in Europe, is FAME. However, ethanol can also be used as thealcohol in transesterification, in which case the biodiesel is fattyacid ethyl ester (FAEE). In the US, the two types are usually notdistinguished, and are collectively known as fatty acid alkyl esters(FAAE), which as a generic term can apply regardless of the acylacceptor used. Direct hydrogenation can also be utilized to convert atleast a portion of the algal oil to a biodiesel. As such, in one aspect,the biodiesel product can include an alkane.

The algal triglyceride can also be converted to biodiesel by directhydrogenation. In this process, the products are alkane chains, propane,and water. The glycerol backbone is hydrogenated to propane, so there issubstantially no glycerol produced as a byproduct. Furthermore, noalcohol or transesterification catalysts are needed. All of the biomasscan be used as feed for the oil-producing algae with none needed forfermentation to produce alcohol for transesterification. The resultingalkanes are pure hydrocarbons, with no oxygen, so the biodiesel producedin this way has a slightly higher energy content than the alkyl esters,degrades more slowly, does not attract water, and has other desirablechemical properties.

Feedstuffs

The present invention includes compositions which can be used asfeedstuffs. For purposes of the present invention, “feedstuffs” includeany food or preparation for human or animal consumption (including forenteral and/or parenteral consumption) which when taken into the body:(1) serve to nourish or build up tissues or supply energy, and/or (2)maintain, restore or support adequate nutritional status or metabolicfunction. Feedstuffs of the invention include nutritional compositionsfor babies and/or young children.

Feedstuffs of the invention comprise for example, a cell of theinvention, a plant of the invention, the plant part of the invention,the seed of the invention, an extract of the invention, the product of amethod of the invention, the product of a fermentation process of theinvention, or a composition along with a suitable carrier(s). The term“carrier” is used in its broadest sense to encompass any component whichmay or may not have nutritional value. As the person skilled in the artwill appreciate, the carrier must be suitable for use (or used in asufficiently low concentration) in a feedstuff, such that it does nothave deleterious effect on an organism which consumes the feedstuff.

The feedstuff of the present invention comprises a lipid produceddirectly or indirectly by use of the methods, cells or organismsdisclosed herein. The composition may either be in a solid or liquidform. Additionally, the composition may include edible macronutrients,vitamins, and/or minerals in amounts desired for a particular use. Theamounts of these ingredients will vary depending on whether thecomposition is intended for use with normal individuals or for use withindividuals having specialized needs such as individuals suffering frommetabolic disorders and the like.

Examples of suitable carriers with nutritional value include, but arenot limited to, macronutrients such as edible fats, carbohydrates andproteins. Examples of such edible fats include, but are not limited to,coconut oil, borage oil, fungal oil, black current oil, soy oil, andmono- and di-glycerides. Examples of such carbohydrates include, but arenot limited to, glucose, edible lactose, and hydrolyzed starch.Additionally, examples of proteins which may be utilized in thenutritional composition of the invention include, but are not limitedto, soy proteins, electrodialysed whey, electrodialysed skim milk, milkwhey, or the hydrolysates of these proteins.

With respect to vitamins and minerals, the following may be added to thefeedstuff compositions of the present invention, calcium, phosphorus,potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc,selenium, iodine, and vitamins A, E, D, C, and the B complex. Other suchvitamins and minerals may also be added.

The components utilized in the feedstuff compositions of the presentinvention can be of semi-purified or purified origin. By semi-purifiedor purified is meant a material which has been prepared by purificationof a natural material or by de novo synthesis.

A feedstuff composition of the present invention may also be added tofood even when supplementation of the diet is not required. For example,the composition may be added to food of any type, including, but notlimited to, margarine, modified butter, cheeses, milk, yogurt,chocolate, candy, snacks, salad oils, cooking oils, cooking fats, meats,fish and beverages.

The genus Saccharomyces spp is used in both brewing of beer and winemaking and also as an agent in baking, particularly bread. Yeast is amajor constituent of vegetable extracts. Yeast is also used as anadditive in animal feed. It will be apparent that genetically modifiedyeast strains can be provided which are adapted to synthesize lipid asdescribed herein. These yeast strains can then be used in food stuffsand in wine and beer making to provide products which have enhancedlipid content.

Additionally, lipid produced in accordance with the present invention orhost cells transformed to contain and express the subject genes may alsobe used as animal food supplements to alter an animal's tissue or milkfatty acid composition to one more desirable for human or animalconsumption. Examples of such animals include sheep, cattle, horses andthe like.

Furthermore, feedstuffs of the invention can be used in aquaculture toincrease the levels of fatty acids in fish for human or animalconsumption.

Preferred feedstuffs of the invention are the plants, seed and otherplant parts such as leaves, fruits and stems which may be used directlyas food or feed for humans or other animals. For example, animals maygraze directly on such plants gown in the field, or be fed more measuredamounts in controlled feeding. The invention includes the use of suchplants and plant parts as feed for increasing the polyunsaturated fattyacid levels in humans and other animals.

Compositions

The present invention also encompasses compositions, particularlypharmaceutical compositions, comprising one or more lipids producedusing the methods of the invention.

A pharmaceutical composition may comprise one or more of the lipids, incombination with a standard, well-known, non-toxicpharmaceutically-acceptable carrier, adjuvant or vehicle such asphosphate-buffered saline, water, ethanol, polyols, vegetable oils, awetting agent, or an emulsion such as a water/oil emulsion. Thecomposition may be in either a liquid or solid form. For example, thecomposition may be in the form of a tablet, capsule, ingestible liquid,powder, topical ointment or cream. Proper fluidity can be maintained forexample, by the maintenance of the required particle size in the case ofdispersions and by the use of surfactants. It may also be desirable toinclude isotonic agents for example, sugars, sodium chloride, and thelike. Besides such inert diluents, the composition can also includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening agents, flavoring agents and perfuming agents.

Suspensions, in addition to the active compounds, may comprisesuspending agents such as ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth,or mixtures of these substances.

Solid dosage forms such as tablets and capsules can be prepared usingtechniques well known in the art. For example, lipid produced inaccordance with the present invention can be tableted with conventionaltablet bases such as lactose, sucrose, and cornstarch in combinationwith binders such as acacia, cornstarch or gelatin, disintegratingagents such as potato starch or alginic acid, and a lubricant such asstearic acid or magnesium stearate. Capsules can be prepared byincorporating these excipients into a gelatin capsule along withantioxidants and the relevant lipid(s).

For intravenous administration, the lipids produced in accordance withthe present invention or derivatives thereof may be incorporated intocommercial formulations.

A typical dosage of a particular fatty acid is from 0.1 mg to 20 g,taken from one to five times per day (up to 100 g daily) and ispreferably in the range of from about 10 mg to about 1, 2, 5, or 10 gdaily (taken in one or multiple doses). As known in the art, a minimumof about 300 mg/day of fatty acid, especially polyunsaturated fattyacid, is desirable. However, it will be appreciated that any amount offatty acid will be beneficial to the subject.

Possible routes of administration of the pharmaceutical compositions ofthe present invention include for example, enteral and parenteral. Forexample, a liquid preparation may be administered orally. Additionally,a homogenous mixture can be completely dispersed in water, admixed understerile conditions with physiologically acceptable diluents,preservatives, buffers or propellants to form a spray or inhalant.

The dosage of the composition to be administered to the subject may bedetermined by one of ordinary skill in the art and depends upon variousfactors such as weight, age, overall health, past history, immunestatus, etc., of the subject.

Additionally, the compositions of the present invention may be utilizedfor cosmetic purposes. The compositions may be added to pre-existingcosmetic compositions, such that a mixture is formed, or a fatty acidproduced according to the invention may be used as the sole “active”ingredient in a cosmetic composition.

Polypeptides

The terms “polypeptide” and “protein” are generally usedinterchangeably.

A polypeptide or class of polypeptides may be defined by the extent ofidentity (% identity) of its amino acid sequence to a reference aminoacid sequence, or by having a greater % identity to one reference aminoacid sequence than to another. The % identity of a polypeptide to areference amino acid sequence is typically determined by GAP analysis(Needleman and Wunsch, 1970; GCG program) with parameters of a gapcreation penalty=5, and a gap extension penalty=0.3. The query sequenceis at least 100 amino acids in length and the GAP analysis aligns thetwo sequences over a region of at least 100 amino acids. Even morepreferably, the query sequence is at least 250 amino acids in length andthe GAP analysis aligns the two sequences over a region of at least 250amino acids. Even more preferably, the GAP analysis aligns two sequencesover their entire length. The polypeptide or class of polypeptides mayhave the same enzymatic activity as, or a different activity than, orlack the activity of, the reference polypeptide. Preferably, thepolypeptide has an enzymatic activity of at least 10% of the activity ofthe reference polypeptide.

As used herein a “biologically active fragment” is a portion of apolypeptide of the invention which maintains a defined activity of afull-length reference polypeptide for example, MGAT activity.Biologically active fragments as used herein exclude the full-lengthpolypeptide. Biologically active fragments can be any size portion aslong as they maintain the defined activity. Preferably, the biologicallyactive fragment maintains at least 10% of the activity of the fulllength polypeptide.

With regard to a defined polypeptide or enzyme, it will be appreciatedthat % identity figures higher than those provided herein will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polypeptide/enzyme comprisesan amino acid sequence which is at least 60%, more preferably at least65%, more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, more preferably at least 93%, more preferably at least 94%, morepreferably at least 95%, more preferably at least 96%, more preferablyat least 97%, more preferably at least 98%, more preferably at least99%, more preferably at least 99.1%, more preferably at least 99.2%,more preferably at least 99.3%, more preferably at least 99.4%, morepreferably at least 99.5%, more preferably at least 99.6%, morepreferably at least 99.7%, more preferably at least 99.8%, and even morepreferably at least 99.9% identical to the relevant nominated SEQ ID NO.

Amino acid sequence mutants of the polypeptides defined herein can beprepared by introducing appropriate nucleotide changes into a nucleicacid defined herein, or by in vitro synthesis of the desiredpolypeptide. Such mutants include for example, deletions, insertions, orsubstitutions of residues within the amino acid sequence. A combinationof deletions, insertions and substitutions can be made to arrive at thefinal construct, provided that the final polypeptide product possessesthe desired characteristics.

Mutant (altered) polypeptides can be prepared using any technique knownin the art, for example, using directed evolution or rathional designstrategies (see below). Products derived from mutated/altered DNA canreadily be screened using techniques described herein to determine ifthey possess fatty acid acyltransferase activity, for example, MGAT,DGAT, or GPAT/phosphatase activity.

In designing amino acid sequence mutants, the location of the mutationsite and the nature of the mutation will depend on characteristic(s) tobe modified. The sites for mutation can be modified individually or inseries for example, by (1) substituting first with conservative aminoacid choices and then with more radical selections depending upon theresults achieved, (2) deleting the target residue, or (3) insertingother residues adjacent to the located site.

Amino acid sequence deletions generally range from about 1 to 15residues, more preferably about 1 to 10 residues and typically about 1to 5 contiguous residues.

Substitution mutants have at least one amino acid residue in thepolypeptide removed and a different residue inserted in its place. Thesites of greatest interest for substitutional mutagenesis include sitesidentified as the active site(s). Other sites of interest are those inwhich particular residues obtained from various strains or species areidentical. These positions may be important for biological activity.These sites, especially those falling within a sequence of at leastthree other identically conserved sites, are preferably substituted in arelatively conservative manner. Such conservative substitutions areshown in Table 1 under the heading of “exemplary substitutions”.

In a preferred embodiment a mutant/variant polypeptide has only, or notmore than, one or two or three or four conservative amino acid changeswhen compared to a naturally occurring polypeptide. Details ofconservative amino acid changes are provided in Table 1. As the skilledperson would be aware, such minor changes can reasonably be predictednot to alter the activity of the polypeptide when expressed in arecombinant cell.

TABLE 1 Exemplary substitutions. Original Exemplary ResidueSubstitutions Ala (A) val; leu; ile; gly Arg (R) lys Asn (N) gln; hisAsp (D) glu Cys (C) ser Gln (Q) asn; his Glu (E) asp Gly (G) pro, alaHis (H) asn; gln Ile (I) leu; val; ala Leu (L) ile; val; met; ala; pheLys (K) arg Met (M) leu; phe Phe (F) leu; val; ala Pro (P) gly Ser (S)thr Thr (T) ser Trp (W) tyr Tyr (Y) trp; phe Val (V) ile; leu; met; phe,alaDirected Evolution

In directed evolution, random mutagenesis is applied to a protein, and aselection regime is used to pick out variants that have the desiredqualities, for example, increased fatty acid acyltransferase activity.Further rounds of mutation and selection are then applied. A typicaldirected evolution strategy involves three steps:

1) Diversification: The gene encoding the protein of interest is mutatedand/or recombined at random to create a large library of gene variants.Variant gene libraries can be constructed through error prone PCR (see,for example, Leung, 1989; Cadwell and Joyce, 1992), from pools of DNaseIdigested fragments prepared from parental templates (Stemmer, 1994a;Stemmer, 1994b; Crameri et al., 1998; Coco et al., 2001) from degenerateoligonucleotides (Ness et al., 2002, Coco, 2002) or from mixtures ofboth, or even from undigested parental templates (Zhao et al., 1998;Eggert et al., 2005; Jézéquek et al., 2008) and are usually assembledthrough PCR. Libraries can also be made from parental sequencesrecombined in vivo or in vitro by either homologous or non-homologousrecombination (Ostermeier et al., 1999; Volkov et al., 1999; Sieber etal., 2001). Variant gene libraries can also be constructed bysub-cloning a gene of interest into a suitable vector, transforming thevector into a “mutator” strain such as the E. coli XL-1 red (Stratagene)and propagating the transformed bacteria for a suitable number ofgenerations. Variant gene libraries can also be constructed bysubjecting the gene of interest to DNA shuffling (i.e., in vitrohomologous recombination of pools of selected mutant genes by randomfragmentation and reassembly) as broadly described by Harayama (1998).

2) Selection: The library is tested for the presence of mutants(variants) possessing the desired property using a screen or selection.Screens enable the identification and isolation of high-performingmutants by hand, while selections automatically eliminate allnonfunctional mutants. A screen may involve screening for the presenceof known conserved amino acid motifs. Alternatively, or in addition, ascreen may involve expressing the mutated polynucleotide in a hostorganism or part thereof and assaying the level of fatty acidacyltransferase activity by, for example, quantifying the level ofresultant product in lipid extracted from the organism or part thereof,and determining the level of product in the extracted lipid from theorganism or part thereof relative to a corresponding organism or partthereof lacking the mutated polynucleotide and optionally, expressingthe parent (unmutated) polynucleotide. Alternatively, the screen mayinvolve feeding the organism or part thereof labelled substrate anddetermining the level of substrate or product in the organism or partthereof relative to a corresponding organism or part thereof lacking themutated polynucleotide and optionally, expressing the parent (unmutated)polynucleotide.

3) Amplification: The variants identified in the selection or screen arereplicated many fold, enabling researchers to sequence their DNA inorder to understand what mutations have occurred.

Together, these three steps are termed a “round” of directed evolution.Most experiments will entail more than one round. In these experiments,the “winners” of the previous round are diversified in the next round tocreate a new library. At the end of the experiment, all evolved proteinor polynucleotide mutants are characterized using biochemical methods.Rational Design

A protein can be designed rationally, on the basis of known informationabout protein structure and folding. This can be accomplished by designfrom scratch (de novo design) or by redesign based on native scaffolds(see, for example, Hellinga, 1997; and Lu and Berry, Protein StructureDesign and Engineering, Handbook of Proteins 2, 1153-1157 (2007)).Protein design typically involves identifying sequences that fold into agiven or target structure and can be accomplished using computer models.Computational protein design algorithms search the sequence-conformationspace for sequences that are low in energy when folded to the targetstructure. Computational protein design algorithms use models of proteinenergetics to evaluate how mutations would affect a protein's structureand function. These energy functions typically include a combination ofmolecular mechanics, statistical (i.e. knowledge-based), and otherempirical terms. Suitable available software includes IPRO (InterativeProtein Redesign and Optimization), EGAD (A Genetic Algorithm forProtein Design), Rosetta Design, Sharpen, and Abalone.

Also included within the scope of the invention are polypeptides definedherein which are differentially modified during or after synthesis forexample, by biotinylation, benzylation, glycosylation, acetylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to an antibody molecule or othercellular ligand, etc. These modifications may serve to increase thestability and/or bioactivity of the polypeptide of the invention.

Identification of Fatty Acid Acyltransferases

In one aspect, the invention provides a method for identifying a nucleicacid molecule encoding a fatty acid acyltransferase having an increasedability to produce MAG, DAG and/or TAG in a cell.

The method comprises obtaining a cell comprising a nucleic acid moleculeencoding a fatty acid acyltransferase operably linked to a promoterwhich is active in the cell. The nucleic acid molecule may encode anaturally occurring fatty acid acyltransferase such as MGAT, GPAT and/orDGAT, or a mutant(s) thereof. Mutants may be engineered using standardprocedures in the art (see above) such as by performing randommutagenesis, targeted mutagenesis, or saturation mutagenesis on knowngenes of interest, or by subjecting different genes to DNA shuffling.For example, a polynucleotide comprising a sequence selected from anyone of SEQ ID NOs:1 to 44 which encodes a MGAT may be mutated and/orrecombined at random to create a large library of gene variants(mutants) using for example, error-prone PCR and/or DNA shuffling.Mutants may be selected for further investigation on the basis that theycomprise a conserved amino acid motif. For example, in the case of acandidate nucleic acid encoding a MGAT, a skilled person may determinewhether it comprises a sequence as provided in SEQ ID NOs:220, 221, 222,223, and/or 224 before testing whether the nucleic acid encodes afunctional MGAT mutant (by for example, transfection into a host cell,such as a plant cell and assaying for fatty acid acyltransferase (i.e.,MGAT) activity as described herein).

Direct PCR sequencing of the nucleic acid or a fragment thereof may beused to determine the exact nucleotide sequence and deduce thecorresponding amino acid sequence and thereby identify conserved aminoacid sequences. Degenerate primers based on conserved amino acidsequences may be used to direct PCR amplification. Degenerate primerscan also be used as probes in DNA hybridization assays. Alternatively,the conserved amino acid sequence(s) may be detected in proteinhybridization assays that utilize for example, an antibody thatspecifically binds to the conserved amino acid sequences(s), or asubstrate that specifically binds to the conserved amino acidsequences(s) such as, for example, a lipid that binds FLXLXXXN (aputative neutral lipid binding domain; SEQ ID NO:224).

In one embodiment, the nucleic acid molecule comprises a sequence ofnucleotides encoding a MGAT. The sequence of nucleotides may i) comprisea sequence selected from any one of SEQ ID NOs:1 to 44, ii) encode apolypeptide comprising amino acids having a sequence as provided in anyone of SEQ ID NOs:45 to 82, or a biologically active fragment thereof,iii) be at least 50% identical to i) or ii), or iv) hybridize to any oneof i) to iii) under stringent conditions. In another or additionalembodiment, the nucleic acid molecule comprises a sequence ofnucleotides encoding one or more conserved DGAT2 and/or MGAT1/2 aminoacid sequences as provided in SEQ ID NOs:220, 221, 222, 223, and 224. Ina preferred embodiment, the nucleic acid molecule comprises a sequenceof nucleotides encoding the conserved amino acid sequences provided inSEQ ID NO:220 and/or SEQ ID NO:224.

In another embodiment, the nucleic acid molecule comprises a sequence ofnucleotides encoding a GPAT, preferably a GPAT which has phosphataseactivity. The sequence of nucleotides may i) comprise a sequenceselected from any one of SEQ ID NOs:84 to 141, ii) encode a polypeptidecomprising amino acids having a sequence as provided in any one of SEQID NOs:144 to 201, or a biologically active fragment thereof, iii) be atleast 50% identical to i) or ii), or iv) hybridize to any one of i) toiii) under stringent conditions. In another or additional embodiment,the nucleic acid molecule comprises a sequence of nucleotides encodingone or more conserved GPAT amino acid sequences as provided in SEQ IDNOs:225, 226, and 227, or a sequence of amino acids which is at least50%, preferably at least 60%, more preferably at least 65% identicalthereto.

In another embodiment, the nucleic acid molecule comprises a sequence ofnucleotides encoding a DGAT2. The sequence of nucleotides may comprisei) a sequence of nucleotides selected from any one of SEQ ID NO:204 to211, ii) encode a polypeptide comprising amino acids having a sequenceas provided in any one of SEQ ID NO:212 to 219, or a biologically activefragment thereof, iii) be at least 50% identical to i) or ii), or iv)hybridize to any one of i) to iii) under stringent conditions. In apreferred embodiment, the DGAT2 comprises a sequence of nucleotides ofSEQ ID NO:204 and/or a sequence of nucleotides encoding a polypeptidecomprising amino acids having a sequence as provided in SEQ ID NO:212.

A cell comprising a nucleic acid molecule encoding a fatty acidacyltransferase operably linked to a promoter which is active in thecell may be obtained using standard procedures in the art such as byintroducing the nucleic acid molecule into a cell by, for example,calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments. Other methods ofcell transformation can also be used and include, but are not limitedto, the introduction of DNA into plants by direct DNA transfer orinjection. Transformed plant cells may also be obtained usingAgrobacterium-mediated transfer and acceleration methods as describedherein.

The method further comprises determining if the level of MAG, DAG and/orTAG produced in the cell is increased when compared to a correspondingcell lacking the nucleic acid using known techniques in the art such asthose exemplified in Example 1. For instance, lipids can be extracted ina chloroform/methanol solution, dried and separated by thin layerchromatography (TLC). Identities of TAG, DAG, MAG, free fatty acid, andother lipids can be verified with internal lipid standards afterstaining with iodine vapor. The resultant chromatograms can analyzedusing a PhosphorImager and the amount of MAG, DAG and TAG quantified onthe basis of the known amount of internal standards, or alternatively,the cells may be fed sn-2 monooleoylglycerol[¹⁴C] or[¹⁴C]glycerol-3-phosphate and associated radioactivity quantitated byliquid scintillation counting (i.e., the amount of labelled MAG, DAG andTAG is quantified).

The method further comprises identifying a nucleic acid moleculeencoding a fatty acid acyltransferase having an increased ability toproduce MAG, DAG and/or TAG in a cell. In a preferred embodiment, thefatty acid acyltransferase catalyzes an enzyme reaction in the MGATpathway. In a further preferred embodiment, DAG is increased via theMGAT pathway (i.e., acylation of MAG with fatty acyl-CoA is catalysed bya MGAT to form DAG). In another or additional embodiment, the substrateMAG is produced by a GPAT which also has phosphatase activity and/or DAGis acylated with fatty acyl-CoA by a DGAT and/or a MGAT having DGATactivity to form TAG.

Gloss

Certain aspects of the invention relate to measuring the glossiness ofvegetative material as a marker for the level of lipid in the material,with higher glossiness levels being associated with higher lipid levels.

The gloss of the vegetative material can be determined using knownprocedures. Glossmeters (reflectometers) provide a quantifiable way ofmeasuring gloss intensity ensuring consistency of measurement bydefining the precise illumination and viewing conditions. Theconfiguration of both illumination source and observation receptionangles allows measurement over a small range of the overall reflectionangle. The measurement results of a glossmeter are related to the amountof reflected light from a black glass standard with a defined refractiveindex. The ratio of reflected to incident light for the specimen,compared to the ratio for the gloss standard, is recorded as glossunits.

The measurement scale, Gloss Units (GU), of a glossmeter is a scalingbased on a highly polished reference black glass standard with a definedrefractive index having a specular reflectance of 100 GU at thespecified angle. This standard is used to establish an upper pointcalibration of 100 with the lower end point established at 0 on aperfectly matt surface. This scaling is suitable for most non-metallicmaterials.

The optimal or expected level of glossiness of vegetative material islikely to vary between plant species. The skilled person can readilyanalyse the lipid content of vegetative material of different plants ofthe invention and identify a suitable pre-determined level of glossinessthat can be used as a standard in the field for assessing the best timeto harvest a vegetative material from a particular plant species.

EXAMPLES Example 1 General Materials and Methods

Expression of Genes in Plant Cells in a Transient Expression System

Genes were expressed in plant cells using a transient expression systemessentially as described by Voinnet et al. (2003) and Wood et al.(2009). Binary vectors containing the coding region to be expressed by astrong constitutive e35S promoter containing a duplicated enhancerregion were introduced into Agrobacterium tumefaciens strain AGL1. Achimeric binary vector, 35S:p19, for expression of the p19 viralsilencing suppressor was separately introduced into AGL1, as describedin WO2010/057246. A chimeric binary vector, 35S:V2, for expression ofthe V2 viral silencing suppressor was separately introduced into AGL1.The recombinant cells were grown to stationary phase at 28° C. in LBbroth supplemented with 50 mg/L kanamycin and 50 mg/L rifampicin. Thebacteria were then pelleted by centrifugation at 5000 g for 5 min atroom temperature before being resuspended to OD600=1.0 in aninfiltration buffer containing 10 mM MES pH 5.7, 10 mM MgCl₂ and 100 uMacetosyringone. The cells were then incubated at 28° C. with shaking for3 hours after which the OD600 was measured and a volume of each culture,including the viral suppressor construct 35S:p19 or 35S:V2, required toreach a final concentration of OD600=0.125 added to a fresh tube. Thefinal volume was made up with the above buffer. Leaves were theninfiltrated with the culture mixture and the plants were typically grownfor a further three to five days after infiltration before leaf discswere recovered for either purified cell lysate preparation or totallipid isolation.

Purified Leaf Lysate Assay

Nicotiana benthamiana leaf tissues previously infiltrated as describedabove were ground in a solution containing 0.1 M potassium phosphatebuffer (pH 7.2) and 0.33 M sucrose using a glass homogenizer. Leafhomogenate was centrifuged at 20,000 g for 45 minutes at 4° C. afterwhich each supernatant was collected. Protein content in eachsupernatant was measured according to Bradford (1976) using a Wallac1420 multi-label counter and a Bio-Rad Protein Assay dye reagent(Bio-Rad Laboratories, Hercules, Calif. USA). Acyltransferase assaysused 100 μg protein according to Cao et al. (2007) with somemodifications. The reaction medium contained 100 mM Tris-HCl (pH 7.0), 5mM MgCl₂, 1 mg/mL BSA (fatty acid-free), 200 mM sucrose, 40 mM coldoleoyl-CoA, 16.4 μM sn-2 monooleoylglycerol[¹⁴C] (55 mCi/mmol, AmericanRadiochemicals, Saint Louis, Mo. USA) or 6.0 μM[¹⁴C]glycerol-3-phosphate (G-3-P) disodium salt (150 mCi/mmol, AmericanRadiochemicals). The assays were carried out for 7.5, 15, or 30 minutes.

Lipid Analysis

In summary, the methods used for analysing lipids in seeds or vegetativetissues were as follows:

Arabidopsis seed and any other similar sized seed:

(i) Fatty acid composition—direct methylation of fatty acids in seeds,without crushing of seeds.

(ii) Total fatty acid or TAG quantitation—direct methylation of fattyacids in seeds, without crushing of seeds, with use of a 17:0 TAGstandard.

Canola seed, Camelina seed, and any other larger sized seeds:

(i) Single seed fatty acid composition—direct methylation of fatty acidsin seed after breaking seed coat.

(ii) Pooled seed-fatty acid composition of total extractedlipid—crushing seeds in CHCl₃/MeOH and methylation of aliquots of theextracted lipid.

(iii) Pooled seed-total lipid content (seed oil content)—two times lipidextraction for complete recovery of seed lipids after crushing seedsfrom known amount of dessicated seeds, with methylation of lipids fromknown amount of seeds together with 17:0 fatty acids as internalstandard.

(iv) Pooled seed-purified TAG quantitation—two times lipid extractionfor complete recovery of seed lipids after crushing seeds, from knownamount of dessicated seeds, TAG fractionation from the lipid using TLC,and direct methylation of TAG in silica using 17:0 TAG as internalstandard.

Leaf Samples:

(i) Fatty acid composition of total lipid—direct methylation of fattyacids in freeze-dried samples.

(ii) Total lipid quantitation—direct methylation of fatty acids in knownweight of freeze-dried samples, with 17:0 FFA.

(iii) TAG quantitation—because of the presence of substantial amounts ofpolar lipids in leaves, TAG was fractionated by TLC from extracted totallipids, and methylated in the presence of 17:0 TAG internal standard.Steps: Freeze dry samples, weighing, lipid extraction, fractionation ofTAG from known amount of total lipids, direct methylation of TAG insilica together with 17:0 TAG as internal standard.

The methods are detailed as follows:

Analysis of oil content in Arabidopsis seeds

Where seed oil content was to be determined in small seeds such asArabidopsis seeds, seeds were dried in a desiccator for 24 hours andapproximately 4 mg of seed was transferred to a 2 ml glass vialcontaining Teflon-lined screw cap. 0.05 mg triheptadecanoin dissolved in0.1 ml toluene was added to the vial as internal standard. Seed FAMEwere prepared by adding 0.7 ml of 1N methanolic HCl (Supelco) to thevial containing seed material. Crushing of the seeds was not necessarywith small seeds such as Arabidopsis seeds. The mixture was vortexedbriefly and incubated at 80° C. for 2 hours. After cooling to roomtemperature, 0.3 ml of 0.9% NaCl (w/v) and 0.1 ml hexane was added tothe vial and mixed well for 10 minutes in a Heidolph Vibramax 110. TheFAME was collected into a 0.3 ml glass insert and analysed by GC with aflame ionization detector (FID) as mentioned earlier.

The peak area of individual FAME were first corrected on the basis ofthe peak area responses of a known amount of the same FAMEs present in acommercial standard GLC-411 (NU-CHEK PREP, INC., USA). GLC-411 containsequal amounts of 31 fatty acids (% by weight), ranging from C8:0 toC22:6. In case of fatty acids which were not present in the standard,the peak area responses of the most similar FAME was taken. For example,the peak area response of FAMEs of 16:1d9 was used for 16:1d7 and theFAME response of C22:6 was used for C22:5. The corrected areas were usedto calculate the mass of each FAME in the sample by comparison to theinternal standard mass. Oil is stored mainly in the form of TAG and itsweight was calculated based on FAME weight. Total moles of glycerol wasdetermined by calculating moles of each FAME and dividing total moles ofFAMEs by three. TAG was calculated as the sum of glycerol and fatty acylmoieties using a relation: % oil by weight=100×((41× total molFAME/3)+(total g FAME−(15× total mol FAME)))/g seed, where 41 and 15 aremolecular weights of glycerol moiety and methyl group, respectively.

Analysis of Oil Content in Camelina Seeds and Canola Seeds by Extraction

After harvest at plant maturity, Camelina or canola seeds weredessicated by storing the seeds for 24 hours at room temperature in adessicator containing silica gel as dessicant. Moisture content of theseeds is typically 6-8%. Total lipids were extracted from known weightsof the dessicated seeds by crushing the seeds using a mixture ofchloroform and methanol (2/1 v/v) in an eppendorf tube using a Reichttissue layer (22 frequency/seconds for 3 minutes) and a metal ball. Onevolume of 0.1M KCl was added and the mixture shaken for 10 minutes. Thelower non-polar phase was collected after centrifuging the mixture for 5minutes at 3000 rpm. The remaining upper (aqueous) phase was washed with2 volumes of chloroform by mixing for 10 minutes. The second non-polarphase was also collected and pooled with the first. The solvent wasevaporated from the lipids in the extract under nitrogen flow and thetotal dried lipid was dissolved in a known volume of chloroform.

To measure the amount of lipid in the extracted material, a known amountof 17:0-TAG was added as internal standard and the lipids from the knownamount of seeds incubated in 1 N methanolic-HCl (Supelco) for 2 hours at80° C. FAME thus made were extracted in hexane and analysed by GC.Individual FAMEs were quantified on the basis of the amount of 17:0TAG-FAME. Individual FAMEs weights, after subtraction of weights of theesterified methyl groups from FAME, were converted into moles bydividing by molecular weights of individual FAMEs. Total moles of allFAMEs were divided by three to calculate moles of TAG and thereforeglycerol. Then, moles of TAG were converted in to weight of TAG.Finally, the percentage oil content on a seed weight basis wascalculated using seed weights, assuming that all of the extracted lipidis TAG or equivalent to TAG for the purpose of calculating oil content.This method was based on Li et al., (2006). Seeds other than Camelina orcanola seeds that are of a similar size can also be analysed by thismethod.

Canola and other seed oil content can also be measured by nuclearmagnetic resonance techniques (Rossell and Pritchard, 1991), forexample, by a pulsed wave NMS 100 Minispec (Bruker Pty Ltd ScientificInstruments, Germany), or by near infrared reflectance spectroscopy suchas using a NIRSystems Model 5000 monochromator. The NMR method cansimultaneously measure moisture content. Moisture content can also bemeasured on a sample from a batch of seeds by drying the seeds in thesample for 18 hours at about 100° C., according to Li et al., (2006).

Where fatty acid composition is to be determined for the oil in canolaseed, the direct methylation method used for Arabidopsis seed (above)can be used, modified with the addition of cracking of the canolaseedcoat. This method extracts sufficient oil from the seed to allowfatty acid composition analysis.

Analysis of Lipids from Leaf Lysate Assays

Lipids from the lysate assays were extracted usingchloroform:methanol:0.1 M KCl (2:1:1) and recovered. The different lipidclasses in the samples were separated on Silica gel 60 thin layerchromatography (TLC) plates (MERCK, Dermstadt, Germany) impregnated with10% boric acid. The solvent system used to fractionate TAG from thelipid extract consisted of chloroform/acetone (90/10 v/v). Individuallipid classes were visualized by exposing the plates to iodine vapourand identified by running parallel authentic standards on the same TLCplate. The plates were exposed to phosphor imaging screens overnight andanalysed by a Fujifilm FLA-5000 phosphorimager before liquidscintillation counting for DPM quantification.

Total Lipid Isolation and Fractionation

Tissues including leaf samples were freeze-dried, weighed (dry weight)and total lipids extracted as described by Bligh and Dyer (1959) or byusing chloroform:methanol:0.1 M KCl (CMK; 2:1:1) as a solvent. Totallipids were extracted from N. benthamiana leaf samples, after freezedying, by adding 900 μL of a chloroform/methanol (2/1 v/v) mixture per 1cm diameter leaf sample. 0.8 μg DAGE was added per 0.5 mg dry leafweight as internal standard when TLC-FID analysis was to be performed.Samples were homogenized using an IKA ultra-turrax tissue lyser afterwhich 500 μL 0.1 M KCl was added. Samples were vortexed, centrifuged for5 min and the lower phase was collected. The remaining upper phase wasextracted a second time by adding 600 μL chloroform, vortexing andcentrifuging for 5 min. The lower phase was recovered and pooled intothe previous collection. Lipids were dried under a nitrogen flow andresuspended in 2 μL chloroform per mg leaf dry weight. Total lipids ofN. tabacum leaves or leaf samples were extracted as above with somemodifications. If 4 or 6 leaf discs (each approx 1 cm² surface area)were combined, 1.6 ml of CMK solvent was used, whereas if 3 or less leafdiscs were combined, 1.2 ml CMK was used. Freeze dried leaf tissues werehomogenized in an eppendorf tube containing a metallic ball using aReicht tissue lyser (Qiagen) for 3 minutes at 20 frequency/sec.

Separation of Neutral Lipids Via TLC and Transmethylation

Known volumes of total leaf extracts such as, for example, 30 μL, wereloaded on a TLC silica gel 60 plate (1×20 cm) (Merck KGaA, Germany). Theneutral lipids were separated via TLC in an equilibrated developmenttank containing a hexane/DEE/acetic acid (70/30/1 v/v/v/) solventsystem. The TAG bands were visualised by iodine vapour, scraped from theTLC plate, transferred to 2 mL GC vials and dried with N₂. 750 μL of 1Nmethanolic-HCl (Supelco analytical, USA) was added to each vial togetherwith a known amount of C17:0 TAG, such as, for example, 30 μg, asinternal standard for quantification.

When analysing the effect on oleic acid levels of specific genecombinations, TAG and polar lipids bands were collected from the TLCplates. Next, 15 μg of C17:0 internal standard was added to samples suchas TAG samples, polar lipid samples and 20 μL of the total lipidextracts. Following drying under N₂, 70 μL toluene and 700 μL methanolicHCl were added.

Lipid samples for fatty acid composition analysis by GC weretransmethylated by incubating the mixtures at 80° C. for 2 hours in thepresence of the methanolic-HCl. After cooling samples to roomtemperature, the reaction was stopped by adding 350 μl H₂O. Fatty acylmethyl esters (FAME) were extracted from the mixture by adding 350 μlhexane, vortexing and centrifugation at 1700 rpm for 5 min. The upperhexane phase was collected and transferred into GC vials with 300 μlconical inserts. After evaporation, the samples were resuspended in 30μl hexane. One μl was injected into the GC.

The amount of individual and total fatty acids (TFA) present in thelipid fractions was quantified by GC by determining the area under eachpeak and calculated by comparison with the peak area for the knownamount of internal standard. TAG content in leaf was calculated as thesum of glycerol and fatty acyl moieties in the TAG fraction using arelation: % TAG by weigh=100×((41× total mol FAME/3)+(total g FAME−(15×total mol FAME)))/g leaf dry weight, where 41 and 15 are molecularweights of glycerol moiety and methyl group, respectively.

Capillary Gas-Liquid Chromatography (GC)

FAME were analysed by GC using an Agilent Technologies 7890A GC (PaloAlto, Calif., USA) equipped with an SGE BPX70 (70% cyanopropylpolysilphenylene-siloxane) column (30 m×0.25 mm i.d., 0.25 μm filmthickness), an FID, a split/splitless injector and an AgilentTechnologies 7693 Series auto sampler and injector. Helium was used asthe carrier gas. Samples were injected in split mode (50:1 ratio) at anoven temperature of 150° C. After injection, the oven temperature washeld at 150° C. for 1 min, then raised to 210° C. at 3° C. min⁻¹ andfinally to 240° C. at 50° C. min⁻¹ Peaks were quantified with AgilentTechnologies ChemStation software (Rev B.04.03 (16), Palo Alto, Calif.,USA) based on the response of the known amount of the external standardGLC-411 (Nucheck) and C17:0-Me internal standard.

Quantification of TAG Via Iatroscan

One μL of lipid extract was loaded on one Chromarod-SII for TLC-FIDIatroscan™ (Mitsubishi Chemical Medience Corporation—Japan). TheChromarod rack was then transferred into an equilibrated developing tankcontaining 70 mL of a hexane/CHCl₃/2-propanol/formic acid(85/10.716/0.567/0.0567 v/v/v/v) solvent system. After 30 min ofincubation, the Chromarod rack was dried for 3 min at 100° C. andimmediately scanned on an Iatroscan MK-6s TLC-FID analyser (MitsubishiChemical Medience Corporation—Japan). Peak areas of DAGE internalstandard and TAG were integrated using SIC-48011 integration software(Version: 7.0-E SIC System instruments Co., LTD—Japan).

TAG quantification was carried out in two steps. First, DAGE was scannedin all samples to correct the extraction yields after which concentratedTAG samples were selected and diluted. Next, TAG was quantified indiluted samples with a second scan according to the external calibrationusing glyceryl trilinoleate as external standard (Sigma-Aldrich).

Quantification of TAG in Leaf Samples by GC

The peak area of individual FAME were first corrected on the basis ofthe peak area responses of known amounts of the same FAMEs present in acommercial standard GLC-411 (NU-CHEK PREP, Inc., USA). The correctedareas were used to calculate the mass of each FAME in the sample bycomparison to the internal standard. Since oil is stored primarily inthe form of TAG, the amount of oil was calculated based on the amount ofFAME in each sample. Total moles of glycerol were determined bycalculating the number of moles of FAMEs and dividing total moles ofFAMEs by three. The amount of TAG was calculated as the sum of glyceroland fatty acyl moieties using the formula: % oil by weight=100×((41×total mol FAME/3)+(total g FAME−(15× total mol FAME)))/g leaf dryweight, where 41 and 15 were the molecular weights of glycerol moietyand methyl group, respectively.

DGAT Assay in Saccharomyces cerevisiae H1246

Saccharomyces cerevisiae strain H1246 is completely devoid of DGATactivity and lacks TAG and sterol esters as a result of knockoutmutations in four genes (DGA1, LRO1, ARE1, ARE2). The addition of freefatty acid (e.g. 1 mM 18:1^(Δ9)) to H1246 growth media is toxic in theabsence of DGAT activity. Growth on such media can therefore be used asan indicator or selection for the presence of DGAT activity in thisyeast strain.

S cerevisiae H1246 was transformed with the pYES2 construct (negativecontrol), a construct encoding Arabidopsis thaliana DGAT1 in pYES2, or aconstruct encoding Mus musculus MGAT2 in pYES2. Transformants were fed[¹⁴C]18:1^(Δ9) free fatty acids.

In a separate experiment, S cerevisiae H1246 was transformed with thepYES2 construct (negative control), a construct encoding Bernadiapulchella DGAT1 in pYES2, or a construct encoding M. musculus MGAT1 inpYES2 and fed 18:1^(Δ9) free fatty acids. S. cerevisiae S288C wild typestrain transformed with pYES2 served as a positive control.

Yeast transformants were resuspended in sterile mQ water and diluted toOD600=1. Samples were further diluted in four consecutive dilutions,each at 1/10. 2 μl of each dilution was spotted on each of the plates(YNBD, YNBG, YNBG+FA) together with 2 μL mQ water and 2 μL of anuntransformed H1246 cell suspension (OD600=1). Plates were incubated for6 days at 30° C. before scoring growth.

Plate Medium, 40 mL Media Per Plate

-   -   YNBD: minimal dropout medium lacking uracil and containing 2%        glucose, 0.01% NP40 and 100 μL ethanol.    -   YNBG: minimal dropout medium lacking uracil and containing 2%        galactose, 1% raffinose, 0.01% NP40 and 100 μL ethanol.    -   YNBG+FA: minimal dropout medium lacking uracil and containing 2%        galactose, 1% raffinose, 0.01% NP40 and 1 mM C18:1^(Δ9)        dissolved in 100 μl ethanol.

Example 2 Constitutive Expression of a Monoacylglycerol Acyltransferasein Plant Cells

MGAT1

The enzyme activity of the monoacylglycerol acyltransferase 1 (MGAT1)encoded by the gene from M. musculus (Yen et al., 2002) and A. thalianadiacylglycerol acyltransferase (DGAT1) (Bouvier-Nave et al., 2000), usedhere as a comparison with MGAT1, were demonstrated in N. benthamianaleaf tissue using a transient expression system as described in Example1.

A vector designated 35S-pORE04 was made by inserting a PstI fragmentcontaining a 35S promoter into the SfoI site of vector pORE04 after T4DNA polymerase treatment to blunt the ends (Coutu et al., 2007). Achimeric DNA encoding the M. musculus MGAT1, codon-optimised forBrassica napus, was synthesized by Geneart and designated0954364_MGAT_pMA. A chimeric DNA designated 35S:MGAT1 and encoding theM. musculus MGAT1 (Genbank Accession No. Q91ZV4) for expression in plantcells was made by inserting the entire coding region of0954364_MGAT_pMA, contained within an EcoRI fragment, into 35S-pORE04 atthe EcoRI site. The vector containing the 35S:MGAT1 construct wasdesignated as pJP3184. Similarly, a chimeric DNA 35S:DGAT1 encoding theA. thaliana DGAT1 (Genbank Accession No. AAF19262) for expression inplant cells was made by inserting the entire coding region of pXZP513E,contained within a BamHI-EcoRV fragment, into 35S-pORE04 at theBamHI-EcoRV site. The vector containing the 35S:DGAT1 construct wasdesignated pJP2078.

The chimeric vectors were introduced into A. tumefaciens strain AGL1 andcells from cultures of these infiltrated into leaf tissue of N.benthamiana plants in a 24° C. growth room. In order to allow directcomparisons between samples and to reduce inter-leaf variation, samplesbeing compared were infiltrated on either side of the same leaf.Experiments were performed in triplicate. Following infiltration, theplants were grown for a further three days before leaf discs were taken,freeze-dried, and lipids extracted from the samples were fractionatedand quantified as described in Example 1. This analysis revealed thatthe MGAT1 and DGAT1 genes were functioning to increase leaf oil levelsin N. benthamiana as follows.

Leaf tissue transformed with the 35S:p19 construct only (negativecontrol) contained an average of 4 μg free fatty acid (FFA) derived fromDAG/mg dry leaf weight and 5 μg FFA derived from TAG/mg dry leaf weight.Leaf tissue transformed with the 35S:p19 and 35S:DGAT1 constructs(control for expression of DGAT1) contained an average of 4 μg FFAderived from DAG/mg dry leaf weight and 22 μg FM derived from TAG/mg dryleaf weight. Leaf tissue transformed with the 35S:p19 and 35S:MGAT1constructs contained an average of 8 μg FFA derived from DAG/mg dry leafweight and 44 μg FFA derived from TAG/mg dry leaf weight. Leaf tissuetransformed with the 35S:p19, 35S:DGAT1 and 35S:MGAT1 constructs did notcontain DAG or TAG levels higher than those observed in the 35S:p19 and35S:MGAT1 infiltration (FIG. 2). Also, a decrease in the level ofsaturates in seeds was noted after MGAT expression when compared witheither the p19 control or DGAT1 samples (Table 2).

The data described above demonstrated that the MGAT1 enzyme was far moreactive than the DGAT1 enzyme in promoting both DAG and TAG accumulationin leaf tissue. Expression of the MGAT1 gene resulted in twice as muchTAG and DAG accumulation in leaf tissue compared to when the DGAT1 wasexpressed. This result was highly surprising and unexpected, consideringthat the MGAT is an enzyme expressed in mouse intestine, a vastlydifferent biological system than plant leaves. This study was the firstdemonstration of ectopic MGAT expression in a plant cell.

Leaf samples infiltrated with M. musculus MGAT1 accumulated double theDAG and TAG relative to leaf tissue infiltrated with A. thaliana DGAT1alone. The efficiency of the production of TAG was also surprising andunexpected given that the mouse MGAT has only very low activity as aDGAT. Leaf tissue infiltrated with genes encoding both MGAT1 and DGAT1did not accumulate significantly more TAG than the MGAT1-only leafsample. FIG. 1 is a representation of various TAG accumulation pathways,most of which converge at DAG, a central molecule in lipid synthesis.For instance, MAG, DAG and TAG can be inter-converted via various enzymeactivities including transacylation, lipase, MGAT, DGAT and PDAT. Adecrease in the level of saturates was also noted after MGAT expression.

MGAT2

A chimeric DNA designated 35S:MGAT2 and encoding the M. musculus MGAT2for expression in plant cells was made by inserting the entire MGAT2coding region, contained within an EcoRI fragment, into 35S-pORE04 atthe EcoRI site. The enzyme activity of the monoacylglycerolacyltransferase 2 (MGAT2) encoded by the gene from M. musculus (Yen,2003) (Genbank Accession No. Q80W94) and A. thaliana DGAT1 (Bouvier-Naveet al., 2000), used here as a comparison with MGAT2, was alsodemonstrated in N. benthamiana leaf tissue using a transient expressionsystem as described in Example 1.

Compared with controls, DGAT1 expression increased leaf TAG 5.9-fold,MGAT2 by 7.3-fold and the combination of MGAT2+DGAT1 by 9.8-fold (FIG.3). The ability of MGAT2 alone to yield such significant increases inTAG was unexpected for a number of reasons. Firstly, the amount ofsubstrate MAG present in leaf tissue is known to be low and largeincreases in TAG accumulation from this substrate would not be expected.Secondly, the addition of MGAT activity alone (i.e., addition of MGAT2which does not have DGAT activity) would be expected to yield DAG, notTAG, especially in a leaf environment where little native DGAT activityis usually present.

Discussion

The present inventors have surprisingly demonstrated that the transgenicexpression of a MGAT gene results in significant increases in lipidyield in plant cells. The present inventors understand that Tumaney etal. had isolated a DGAT with some MGAT activity and that they were notsuccessful in attempts to clone a gene encoding a MGAT as definedherein. Tumaney et al. (2001) reported MGAT activity in peanut andisolated an enzyme responsible for this activity. However, Tumaney etal. did not publish results of tests for DGAT activity and it thereforeseems that the enzyme reported was a DGAT with some MGAT activity.Indeed, previous work had failed to identify any MGAT activity in otherspecies (Stobart et al., 1997). Furthermore, it was surprising that theenzyme isolated by Tumaney et al. was a soluble, cytosolic, enzymerather than a membrane-bound enzyme.

Recently, researchers have identified a microsomal membrane-boundmonoacylglycerol acyltransferase (MGAT) from immature peanut (Arachishypogaea) seeds. The MGAT could be solubilized from microsomal membranesusing a combination of a chaotropic agent and a zwitterionic detergent,and a functionally active 14S multiprotein complex was isolated andcharacterized. Oleosin3 (OLE3) was identified as part of themultiprotein complex, which is capable of performing bifunctionalactivities such as acylating monoacylglycerol (MAG) to diacylglycerol(DAG) and phospholipase A2 (PLA2; Parthibane et al., 2012).

Example 3 Biochemical Demonstration of Transgenic MGAT Activity in LeafExtracts

Cell lysates were made from N. benthamiana leaf tissue that had beeninfiltrated with 35S:MGAT1, 35S:MGAT2 and 35S:DGAT1, as described inExample 1. Separate leaf infiltrations were performed, each intriplicate, for strains containing the 35S:p19 construct only (negativecontrol), the 35S:MGAT2 strain together with the 35S:p19 strain, and amixture of the 35S:MGAT2 and 35S:DGAT1 Agrobacterium strains with the35S:p19 strain. The triplicate samples were harvested after three daysand a purified cell lysate prepared by mechanical tissue lysis andcentrifugation. The MGAT activities of the purified cell lysates werecompared by feeding [¹⁴C]MAG to the lysates as described in Example 1.The data are shown in FIG. 4.

Little MGAT activity was observed in the 35S:p19 control sample, sincemost of the radioactivity remained in MAG throughout the assay. Incontrast, the majority of the labelled MAG in the 35S:MGAT2 sample wasrapidly converted to DAG (FIG. 4, central panel), indicating strong MGATactivity expressed from the 35S:MGAT2 construct. Furthermore, asignificant amount of TAG was also produced. The TAG production observedin the 35S:MGAT2 sample was likely due to native N. benthamiana DGATactivity, or produced by another TAG synthesis route. The amount of TAGproduction was greatly increased by the further addition of 35S:DGAT1(FIG. 4, right hand panel), indicating that the MGAT2 enzyme producedDAG which was accessible for conversion to TAG by DGAT1 in plantvegetative tissues.

Example 4 Biochemical Demonstration of the Production of MGAT-AccessibleMAG in Leaf Extracts

In the in vitro assays described in Example 3 using leaf lysates, thesubstrates (sn-2 MAG and oleoyl-CoA) were exogenously supplied, whereasin vivo MGAT activity in intact plant tissues would require the nativepresence of these substrates. The presence of low levels of MAG isvarious plant tissues has been reported previously (Hirayama and Hujii,1965; Panekina et al., 1978; Lakshminarayana et al., 1984; Perry &Harwood, 1993). To test whether the MGAT2 could access MAG produced bynative plant pathways, the above experiment was repeated but this timefeeding [¹⁴C]G-3-P to the lysates. The resultant data are shownschematically in FIG. 5.

The production of labelled MAG was observed in all samples, indicatingthe de novo production of MAG from the G-3-P in plant leaf lysates.Labelled DAG and TAG products were also observed in all samples althoughthese were relatively low in the 35S:p19 control sample, indicating thatthe production of these neutral lipids by the endogenous Kennedy pathwaywas relatively low in this sample. In contrast, the majority of thelabel in the MGAT2 and MGAT2+DGAT1 samples appeared in the DAG and TAGpools, indicating that the exogenously added MGAT catalysed conversionof the MAG that had been produced from the labelled G-3-P by a nativeplant pathway.

Examples 2 to 4 demonstrate several key points: 1) Leaf tissue cansynthesise MAG from G-3-P such that the MAG is accessible to anexogenous MGAT expressed in the leaf tissue; 2) Even an MGAT which isderived from mammalian intestine can function in plant tissues, notknown to possess an endogenous MGAT, requiring a successful interactionwith other plant factors involved in lipid synthesis; 3) DAG produced bythe exogenous MGAT activity is accessible to a plant DGAT, or anexogenous DGAT, to produce TAG; and 4) the expression of an exogenousMGAT can yield greatly increased TAG levels in plant tissues, levelswhich are at least as great as that yielded by exogenous A. thalianaDGAT1 expression.

Example 5 Expression of DGAT1, MGAT1 and MGAT2 in Yeast

Chimeric yeast expression vectors were constructed by inserting genesencoding the A. thaliana DGAT1, M. musculus MGAT1 and M. musculus MGAT2into the pYES2 vector to yield pYES2:DGAT1, pYES2:MGAT1 and pYES2:MGAT2.These constructs were transformed in Saccharomyces cerevisiae strainH1246 which is completely devoid of DGAT activity and lacks TAG andsterol esters as a result of knockout mutations in four genes (DGA1,LRO1, ARE1, ARE2). Yeast strain H1246 is capable of synthesizing DAGfrom exogenously added fatty acids, but is unable to convert the DAG toTAG because of the knockout mutations. The transformed yeast cultureswere fed [¹⁴C]18:1^(Δ9) before total lipids were extracted andfractionated by TLC as described in Example 1. An autoradiogram of arepresentative TLC plate is shown in FIG. 6.

TAG formation, indicating the presence of DGAT activity, was observedfor the yeast cells containing either DGAT1 (positive control) and themammalian MGAT1, but not in cells containing the MGAT2 encoded by thenative M. musculus coding region. It was concluded that MGAT1 from mousealso had DGAT activity in yeast cells, and therefore functioned as adual function MGAT/DGAT enzyme, whereas MGAT2 did not have detectableDGAT activity and was therefore solely an MGAT. A construct whichincluded an MGAT2 coding region which was codon optimization forexpression in yeast exhibited MGAT activity (production of DAG) whentested in vitro using yeast microsomes and labelled MAG substrate,whereas a similar construct which was codon-optimised for expression inB. napus did not show DAG production in the yeast microsomes. Thisexperiment showed the benefit of codon-optimisation for the organism inwhich heterologous coding regions were to be expressed.

Example 6 Expression of a Monoacylglycerol Acyltransferase in Plants,Seeds and Fungi

Expression of MGAT1 in Arabidopsis thaliana Seeds

A gene encoding M. musculus MGAT1 and under the control of aseed-specific promoter (FP1, a truncated Brassica napus napin promoter)was used to generate stably transformed A. thaliana plants and progenyseeds. The vector designated pJP3174 was made by inserting a SalIfragment containing an EcoRI site flanked by the FP1 promoter andGlycine max lectin polyadenylation signal into the SalI-XhoI site ofvector pCW141. The pCW141 vector also contained an FP1-driven,intron-interrupted, seed-secreted GFP as a screenable marker gene. Thechimeric gene designated FP1:MGAT1-GFP was made by inserting the entirecoding region of the construct 0954364_MGAT_pMA, contained within anEcoRI fragment, into pJP3174 at the EcoRI site, generating pJP3179. Thischimeric vector was introduced into A. tumefaciens strain AGL1 and cellsfrom culture of the transformed Agrobacterium used to treat A. thaliana(ecotype Columbia) plants using the floral dip method for transformation(Clough and Bent, 1998). After maturation, the seeds from the treatedplants were viewed under a Leica MZFLIII dissection microscope and ebq100 mercury lamp. Fifteen transgenic seeds (strongly GFP positive) andfifteen non-transgenic (GFP negative) seeds were isolated and each setpooled. The GFP positive and GFP negative pools were analysed for totalfatty acid content as described in Example 1. This analysis provided theaverage fatty acid content and composition for seeds transformed withthe MGAT construct, but in a population which may have contained bothhemizygous and homozygous transformed seeds.

This analysis revealed that the MGAT1 gene was functioning to increaseseed oil levels in A. thaliana seed with the fifteen non-transgenicseeds (control, the same as wild-type) containing an average of 69.4 μgtotal fatty acids while the fifteen transgenic seeds transformed withthe GFP gene, and therefore likely to contain the FP1:MGAT1 geneticconstruct, contained an average of 71.9 μg total fatty acids. This wasan increase of 3.5% in the oil content relative to the control(wild-type). The analysis also revealed that the MGAT gene wasfunctioning to enrich polyunsaturated fatty acids in the seed, as seenfrom the fatty acid composition of the total extracted lipid obtainedfrom the seeds. In particular, the amount of ALA present as a percentageof the total fatty acid extracted from the seeds increasing from 16.0 to19.6%. Similarly, the percentage of the fatty acid 20:2n6 increased from1.25% to 1.90% and the fatty acid 20:3n3 increased from 0.26% to 0.51%(Table 2).

TABLE 2 Effect of MGAT expression on seed fatty acid composition. FAprofile (% of TFA) Sample 16:00 16:01 16:3w3 C18:0 C18:1d9 C18:1d11C18:2 C18:3 Control 7.41 0.36 0.12 3.00 15.26 1.98 30.93 15.98 MGAT17.11 0.32 0.11 2.95 13.86 1.51 28.87 19.59 Sample C20:0 20:1d11 20:1iso20:2n6 20:3n3 C22:0 C22:1 C24:0 24:1d15 Total Control 1.86 17.95 1.741.25 0.26 0.57 0.98 0.20 0.17 100.00 MGAT1 1.90 17.22 1.71 1.90 0.510.57 1.52 0.19 0.17 100.00

A further experiment was performed where the FPLMGAT1-GFP chimeric DNAwas modified to remove the GFP gene. This genetic construct, designatedFPLMGAT1, was transformed into an A. thaliana line which was mutant forFAD2. The total fatty acid content of the T₂ seed from antibioticresistant T₁ plants, as well as parental lines grown alongside theseplants, was determined according to Example 1. The data is shown inTable 3. The average total fatty acids of the seed from the controllines was 347.9 μg/100 seeds whereas the average of the transgenic seedswas 381.0 μg/100 seeds. When the data for the control line C6 wasexcluded for determining the average, the average for the controls was370 μg/100 seeds. The oil content in the transgenic seeds represented anincrease of about 3% in relative terms compared to the oil content inthe untransformed seeds.

TABLE 3 Arabidopsis thaliana T₂ FP1:MGAT1 transgenic and parentalcontrol seed fatty acid profiles and total fatty acid quantification. μgFA/100 Sample C16:0 C16:1 C18:0 C18:1 C18:1d11 C18:2 C18:3 C20:0 20:1d11C22:0 C24:0 24:1d15 seeds C7 6.2 0.5 2.5 81.3 4.2 0.6 2.7 0.7 0.7 0.30.2 0.1 442.5 C4 6.3 0.4 2.4 81.7 3.9 0.5 2.5 0.9 0.6 0.4 0.2 0.1 403.8C8 6.4 0.5 2.6 81.1 4.1 0.6 2.6 0.8 0.6 0.4 0.2 0.1 403.2 C2 6.2 0.5 2.481.4 4.1 0.6 2.7 0.8 0.6 0.4 0.2 0.1 377.0 C1 6.4 0.5 2.4 80.6 4.1 0.73.3 0.8 0.6 0.4 0.2 0.1 344.8 C3 6.4 0.5 2.6 80.0 4.1 0.6 3.5 0.8 0.60.4 0.2 0.2 314.3 C5 6.3 0.5 2.6 80.7 4.4 0.6 2.4 0.7 0.6 0.9 0.2 0.1310.6 C6 6.7 0.7 2.7 77.2 5.0 0.8 4.3 0.9 0.7 0.4 0.3 0.2 186.8 M23 5.90.4 2.0 81.4 5.0 0.8 2.4 0.7 0.7 0.5 0.2 0.2 455.7 M10 6.0 0.4 2.4 82.34.2 0.7 2.2 0.7 0.6 0.3 0.2 0.1 437.7 M22 5.9 0.4 2.2 81.4 4.8 0.8 2.40.7 0.6 0.4 0.2 0.2 425.0 M25 6.0 0.4 2.2 81.7 4.6 0.7 2.4 0.8 0.6 0.30.2 0.1 406.7 M8 6.0 0.4 2.2 81.6 4.5 0.8 2.5 0.7 0.6 0.3 0.2 0.2 404.5M14 5.7 0.4 2.1 81.8 4.6 0.8 2.5 0.7 0.6 0.3 0.2 0.2 396.4 M26 6.2 0.42.2 81.8 4.4 0.8 2.2 0.7 0.6 0.4 0.2 0.1 393.0 M6 5.9 0.4 2.2 81.8 4.50.8 2.4 0.7 0.6 0.3 0.2 0.2 392.9 M5 5.9 0.5 2.2 80.9 4.8 0.9 2.6 0.70.7 0.5 0.2 0.2 389.7 M17 6.3 0.4 2.3 75.1 4.7 4.8 4.4 0.7 0.6 0.3 0.20.1 388.4 M9 6.1 0.4 2.3 81.8 4.4 0.7 2.4 0.7 0.6 0.3 0.2 0.1 388.2 M206.2 0.4 2.2 81.5 4.7 0.7 2.3 0.7 0.6 0.3 0.2 0.1 379.1 M12 6.2 0.4 2.281.6 4.4 1.0 2.2 0.7 0.6 0.3 0.2 0.1 374.7 M18 6.2 0.4 2.4 81.3 4.7 0.72.3 0.8 0.5 0.3 0.2 0.1 369.1 M24 6.1 0.4 2.2 81.6 4.6 0.7 2.3 0.9 0.60.3 0.2 0.1 361.7 M7 5.9 0.4 2.3 81.9 4.5 0.7 2.3 0.7 0.6 0.3 0.2 0.1359.4 M4 6.2 0.4 2.4 81.3 4.4 0.7 2.5 0.8 0.5 0.3 0.2 0.2 352.3 M13 6.10.4 2.3 81.4 4.5 0.8 2.4 0.7 0.6 0.3 0.2 0.1 352.0 M16 6.1 0.4 2.2 81.84.4 0.7 2.4 0.7 0.6 0.3 0.2 0.2 340.5 M19 6.1 0.4 2.3 80.9 4.9 0.8 2.70.7 0.5 0.3 0.2 0.1 318.6 M3 6.0 0.5 2.3 81.1 4.7 0.9 2.7 0.7 0.6 0.30.2 0.2 316.6

The coding region of the mouse MGAT2 gene, codon-optimised forexpression in plant cells, was substituted for the MGAT1 coding regionin the constructs mentioned above, and introduced into Arabidopsis.Thirty plants of each transgenic line (T1 and T2 plants, giving rise toT2- and T3-generation seeds) were grown in the greenhouse in a randomlyarranged distribution and compared to control plants. Seeds from thetransgenic plants were increased in their oil content relative to thecontrol seeds (FIG. 7). The average TAG percentage of the T3 transgenicseeds represented a relative increase of about 8% compared to the TAGpercentage in the untransformed seeds (Table 4). A significant increasewas observed in the level of polyunsaturated fatty acids in the TAG ofthe transgenic seeds, in particular of ALA, and a decrease in saturatedfatty acid levels such as palmitic and stearic acids. Moreover, theincreased TAG levels and altered fatty acid composition was morepronounced in the T3 generation than in the T2 seeds, presumably due tothe homozygous state of the transgene in the T3 seeds.

TABLE 4 TAG levels and fatty acid composition in TAG extracted fromArabidopsis thaliana T2 and T3 seeds expressing MGAT2 compared tountransformed control seed. C14:0 C16:0 C16:1 C18:0 C18:1d9 C18:1d11C18:2 C18:3 Control 0.1 8.1 0.3 3.3 13.2 1.8 28.4 20.0 T2 seeds 0.1 7.20.2 2.8 13.0 1.3 27.9 24.3 T3 seeds 0.1 6.1 0.2 2.5 12.3 1.2 28.4 26.7 %TAG by Seed C20:0 C20:1 20:1iso 20:2n6 C22:1 C24:0 24:1 weight Control2.2 17.2 1.9 1.8 1.2 0.3 0.2 32.4 T2 seeds 1.6 14.8 3.1 1.8 1.2 0.3 0.237.9 T3 seeds 1.8 15.3 1.5 1.9 1.5 0.4 0.2 40.2Expression of MGAT1 in Brassica napus Seeds

The vector FP1:MGAT1 used for the expression of M. musculus MGAT1 inArabidopsis thaliana seeds was used to generate transformed B. napusplants. The vector was introduced into A. tumefaciens strain AGL1 viastandard electroporation procedures. Cultures were grown overnight at28° C. in LB medium with agitation at 150 rpm. The bacterial cells werecollected by centrifugation at 4000 rpm for 5 minutes, washed withWinans' AB (Winans, 1988) and re-suspended in 10 mL of Winans' AB medium(pH 5.2) and grown with kanamycin (50 mg/L) and rifampicin (25 mg/L)overnight with the addition of 100 μM acetosyringone. Two hours beforeinfection of the Brassica cells, spermidine (120 mg/L) was added and thefinal density of the bacteria adjusted to an OD 600 nm of 0.3-0.4 withfresh AB media. Freshly isolated cotyledonary petioles from 8-day old B.napus seedlings grown on 1/2 MS (Murashige-Skoog, 1962) or hypocotylsegments preconditioned by 3-4 days on MS media with 1 mg/L thidiazuron(TDZ)+0.1 mg/L alpha-naphthaleneacetic acid (NAA) were infected with 10mL Agrobacterium cultures for 5 minutes. Explants (cotyledonary petioleand hypocotyl) infected with Agrobacterium were then blotted on sterilefilter paper to remove the excess Agrobacterium and transferred toco-cultivation media (MS media with 1 mg/L TDZ+0.1 mg/L NAA+100 μMacetosyringone) supplemented with or without different antioxidants(L-cysteine 50 mg/L and ascorbic 15 mg/L). All the plates were sealedwith parafilm and incubated in the dark at 23-24° C. for 48 hours.

The co-cultivated explants (cotyledonary petiole and hypocotyl) werethen washed with sterile distilled water+500 mg/L cefotaxime+50 mg/Ltimentin for 10 minutes, rinsed in sterile distilled water for 10minutes, blotted dry on sterile filter paper, transferred topre-selection media (MS+1 mg/L TDZ+0.1 mg/L NAA+20 mg/L adenine sulphate(ADS)+1.5 mg/L AgNO₃+250 mg/L cefotaxime and 50 mg/L timentin) andcultured for five days at 24° C. with a 16 hour/8 hour photoperiod. Theywere then transferred to selection media (MS+1 mg/L TDZ+0.1 mg/L NAA+20mg/L ADS+1.5 mg/L AgNO₃+250 mg/L cefotaxime and 50 mg/L timentin) with1.5 mg/L glufosinate ammonium and cultured for 4 weeks at 24° C. with 16hour/8 hour photoperiod with a biweekly subculture onto the same media.Explants with green callus were transferred to shoot initiation media(MS+1 mg/L kinetin+20 mg/L ADS+1.5 mg/L AgNO₃+250 mg/L cefotaxime+50mg/L timentin+1.5 mg/L glufosinate ammonium) and cultured for another2-3 weeks. Shoots emerging form the resistant explants were transferredto shoot elongation media (MS media with 0.1 mg/L gibberelic acid+20mg/L ADS+1.5 mg/L AgNO₃+250 mg/L ceftoxime+1.5 mg/L glufosinate ammoniumand cultured for another two weeks. Healthy shoots 2-3 cm long wereselected and transferred to rooting media (1/2 MS with 1 mg/L NAA+20mg/L ADS+1.5 mg/L AgNO₃+250 mg/L cefotaxime) and cultured for 2-3 weeks.Well established shoots with roots were transferred to pots (seedlingraising mix) and grown in a growth cabinet for two weeks andsubsequently transferred to glasshouse. Sixteen individual transformantsin the cultivar Oscar were confirmed to be transgenic for the FP1:MGAT1construct and grew normally under glasshouse conditions. Plant growthappeared normal and the plants were fertile, flowering and setting seednormally. The plants were grown to maturity and seeds obtained fromtransformed plants were harvested. Seeds from some of the transformedplants were analysed for seed oil content and fatty acid composition.Data from these preliminary analyses showed variability in the oilcontent and fatty acid composition, probably due to the plants beinggrown at different times and under different environmental conditions.To reduce variability, T1 plants which express MGAT1 are produced andgrown under the same conditions as control (wild-type, cultivar Oscar)plants of the same genotype, and the oil content compared.

Expression of MGAT1 in Gossypium hirsutum Seeds

The same seed-specific chimeric gene used for the expression of M.musculus MGAT1 in Arabidopsis thaliana seeds was used to generatetransformed Gossypium hirsutum plants. The vector designated FP1:MGAT1was introduced into A. tumefaciens strain AGL1 via standardelectroporation procedures and cells from the Agrobacterium culture usedto introduce the chimeric DNAs into cells of Gossypium hirsutum, varietyCoker315. Cotyledons excised from 10-day old cotton seedlings were usedas explants and infected and co-cultivated with A. tumefaciens for aperiod of two days. This was followed by a six-week selection on MSmedium (Murashige and Skoog, 1962) containing 0.1 mg/L 2,4-D, 0.1 mg/Lkinetin, 50 mg/L kanamycin sulphate, and 25 mg/L cefotaxime. Healthycalli derived from the cotyledon explants were transferred to MS mediumcontaining 5 mg/L 6-(γ,γ-dimethylallylamino)-purine (2ip), 0.1 mg/Lnaphthalene acetic acid (NAA), 25 mg/L kanamycin, and 250 mg/Lcefotaxime for a second period of six weeks at 28° C. Somatic embryosthat formed after about six to ten weeks of incubation were germinatedand maintained on the same medium, but without added phytohormone orantibiotics. Plantlets developed from the somatic embryos weretransferred to soil and maintained in a glasshouse once leaves and rootswere developed, with 28° C./20° C. (day/night) growth temperature. Tenindependent primary transgenic plants (T0) containing the FP1-MGAT1construct were grown in the glasshouse, flowered and produced bollscontaining seeds. The seeds were harvested on maturity. To enhance thereliability of the oil content analysis, 5 plants were established fromeach of the 10 primary transgenic plants and the mature T2 seeds aresubjected to the analysis of oil content. The seed-specific expressionof MGAT1 increases oil content and increases the percentage ofpolyunsaturated fatty acids in the cotton seedoil.

Expression of a MGAT1 and MGAT2 Genes in N. benthamiana Plants afterStable Transformation

N. benthamiana was stably transformed with the 35S:MGAT1 constructdescribed in Example 2. 35S:MGAT1 was introduced into A. tumefaciensstrain AGL1 via standard electroporation procedure. The transformedcells were grown on solid LB media supplemented with kanamycin (50 mg/L)and rifampicin (25 mg/L) and incubated at 28° C. for two days. A singlecolony was used to initiate fresh culture. Following 48 hours vigorousculture, the cells were collected by centrifugation at 2,000×g and thesupernatant was removed. The cells were resuspended in fresh solutioncontaining 50% LB and 50% MS medium at the density of OD₆₀₀=0.5.

Leaf samples of N. benthamiana grown in vitro were excised and cut intosquare sections around 0.5-1 cm² in size with a sharp scalpel whileimmersed in the A. tumefaciens solution. The wounded N. benthamiana leafpieces submerged in A. tumefaciens were allowed to stand at roomtemperature for 10 minutes prior to being blotted dry on a sterilefilter paper and transferred onto MS plates without supplement.Following a co-cultivation period of two days at 24° C., the explantswere washed three times with sterile, liquid MS medium, then blotted drywith sterile filter paper and placed on the selective MS agarsupplemented with 1.0 mg/L benzylaminopurine (BAP), 0.25 mg/Lindoleacetic acid (IAA), 50 mg/L kanamycin and 250 mg/L cefotaxime. Theplates were incubated at 24° C. for two weeks to allow for shootdevelopment from the transformed N. benthamiana leaf pieces.

To establish rooted transgenic plants in vitro, healthy green shootswere cut off and transferred into 200 mL tissue culture pots containingMS agar medium supplemented with 25 μg/L IAA, 50 mg/L kanamycin and 250mg/L cefotaxime. Sufficiently large leaf discs were taken fromtransgenic shoots and freeze-dried for TAG fractionation andquantification analysis as described in Example 1 (Table 5). The best35S:MGAT1 N. benthamiana plant had a TAG content of 204.85 μg/100 mg dryweight leaf tissue compared with an average TAG content of 85.02 μg/100mg dry weight leaf tissue in the control lines, representing an increasein TAG content of 241%.

N. benthamiana was also stably transformed with the 35S:MGAT2 constructdescribed in Example 2 and a control binary vector pORE4 (Table 6). Thebest 35S:MGAT2 N. benthamiana plant had a TAG content of 79.0 μg/100 mgdry weight leaf tissue compared with a TAG content of 9.5 μg/100 mg dryweight leaf tissue in the control line at the same developmental stage,representing an increase in TAG content of 731%. The fatty acid profileof the TAG fractions was also altered with significantly reduced levelsof the saturated fatty acids 16:0 and 18:0, and increased levels of thepolyunsaturated fatty acids, particularly 18:3ω3 (ALA) (Table 6). Thefatty acid profile of the polar lipids from the same leaf samples werenot significantly affected, indicating that the changes in the fattyacid composition of the non-polar lipids was real. The control plants inthis experiment were smaller and different physiologically than in theprevious experiment with the 35S:MGAT1 construct, and this may haveexplained the different oil contents of the control plants from oneexperiment to the other. Experiments to directly compare the 35S:MGAT1and 35:MGAT2 constructs with control plants are performed using plantsof the same size and physiology.

A new set of constitutive binary expression vectors was made using a 35Spromoter with duplicated enhancer region (e35S). 35S:MGAT1#2 (pJP3346),35S:MGAT2#2 (pJP3347) and 35S:DGAT1#2 (pJP3352) were made by firstcloning the e35S promoter, contained within a BamHI-EcoRI fragment, intopORE04 at the BamHI-EcoRI sites to yield pJP3343. pJP3346 and pJP3347were then produced by cloning the MGAT1 and MGAT2 genes, respectively,into the EcoRI site of pJP3343. pJP3352 was produced by cloning the A.thaliana DGAT1, contained within a XhoI-AsiSI site, into the XhoI-AsiSIsites of pJP3343.

pJP3346, pJP3347 and pJP3352 in Agrobacterium strain AGL1 were used totransform N. benthamiana as described above. Fourteen confirmedtransgenic plants were recovered for pJP3346 and 22 for pJP3347. Anumber of kanamycin resistant, transformed shoots have been generatedwith pJP3352. Expression analysis of the transgenes was performed on theplants transformed with MGAT1 or MGAT2. Plants with high levels ofexpression were selected. Expression analysis on plants transformed withthe A. thaliana DGAT1 is performed. The plants grow normally and aregown to maturity. Seed is harvested when mature. Seed fromhigh-expressing progeny are sown directly onto soil for lipid analysisof the T2 segregating population, which includes both homozygous andheterozygous plants. Oil content of leaves of plants expressing highlevels of either MGAT1 or MGAT2 is significantly increased compared toplants transformed with A. thaliana DGAT1 or control plants. MGAT2transgenic plants showed a significant increase in the unsaturated fattyacid 18:1 and 1% relative increase in total fatty acid content comparedto the null events (Table 7).

pJP3346, pJP3347 and a control vector in AGL1 were also used totransform A. thaliana as described above. Twenty-five confirmedtransgenic T2 plants comprising the T-DNA from pJP3346 and 43 transgenicplants for pJP3347 were identified. Expression analysis was performed onthe transgenic plants. Seeds from high-expressing progeny were harvestedand sown directly onto soil. Lipid analysis including oil content of theleaves from T2 and T3 progeny was performed, including from segregantslacking the transgenes. The highest levels of TAG were obtained inplants that are homozygous for the MGAT transgenes.

TABLE 5 Fatty acid profile and quantification of TAG in Nicotianabenthamiana leaf tissue stably transformed with the 35S:MGAT1 construct.‘M’ samples are 35S:MGAT1 whilst ‘C’ samples are parental controlplants. μg/100 mg Sample C16:0 16:3w3 C18:0 C18:1 C18:1d11 C18:2 C18:3C20:0 20:3n3 C22:0 C24:0 DW M1 38.7 0.7 5.1 8.5 0.4 7.0 34.4 1.1 0.3 0.20.4 204.85 M8 33.2 0.8 4.4 8.1 0.3 6.5 42.8 0.9 0.2 0.2 0.2 184.20 M341.1 0.6 5.3 10.4 0.4 5.5 31.8 1.0 0.4 0.2 0.2 133.62 M2 42.5 0.5 5.27.4 0.0 4.8 34.4 1.0 0.2 0.3 0.2 133.57 M7 35.2 0.6 4.5 8.6 0.0 4.9 41.71.1 0.3 0.3 0.2 128.49 M5 49.1 0.6 6.4 9.0 0.4 3.7 16.9 1.1 0.0 0.5 0.7107.39 M4 41.9 0.4 6.0 9.6 0.0 4.2 33.0 1.1 0.2 0.4 0.2 93.71 M6 41.40.4 5.8 8.2 0.0 4.3 34.6 1.1 0.2 0.3 0.2 88.38 C1 40.2 0.4 6.1 8.3 0.07.8 31.9 1.3 0.2 0.4 0.3 81.53 C2 39.9 0.6 5.5 7.1 0.0 6.9 35.4 1.1 0.30.4 0.3 88.52

TABLE 6 Fatty acid profile and quantification of TAG in Nicotianabenthamiana leaf tissue stably transformed with the 35S:MGAT2 construct.‘M’ samples are 35S:MGAT2 whilst ‘C’ samples are parental controlplants. Two leaves from each plant were taken and analysed separately.Sample C16:0 16:1d7 16:1d13t C16:1 16:3n3 C18:0 C18:1 C18:1d11 C18:2C18:3n C20:0 μg/100 mg DW C, leaf 1 TAG 34.0 2.7 0.8 0.0 0.0 17.3 6.60.0 15.9 18.7 0.0 12.9 C, leaf 2 TAG 35.0 1.8 0.0 0.0 1.3 25.0 3.0 0.013.0 17.6 1.4 6.1 M, leaf 1 TAG 14.6 0.4 1.0 0.4 7.7 5.9 4.0 0.4 16.847.0 0.6 97.1 M, leaf 2 TAG 18.1 0.3 1.0 0.0 6.0 8.1 2.8 0.3 14.0 46.91.0 60.9 C, leaf 1 PL 13.4 0.0 3.0 0.2 7.4 2.0 2.5 0.4 8.4 61.4 0.32439.3 C, leaf 2 PL 10.3 0.0 2.4 0.2 9.7 1.4 2.0 0.3 9.5 63.3 0.0 4811.5M, leaf 1 PL 11.6 0.0 2.4 0.2 8.7 1.9 2.4 0.3 8.7 63.0 0.0 3568.8 M,leaf 2 PL 10.7 0.0 2.4 0.2 9.5 1.6 1.9 0.3 9.2 63.3 0.0 3571.2

TABLE 7 Total fatty acid amount (TFA) and fatty acid composition inNicotiana benthamiana leaf tissues stably transformed with the 35S:MGAT2construct. TFA (ug/100 ug 16:0 16:1 16:3 18:0 18:1d9 18:1d11 18:2 18:3w320:0 20:1 20:1iso 22:0 22:1 24:0 24:1 DW) MGAT 14.5 1.8 5.2 2.1 6.3 0.811.3 53.7 0.4 0.5 0.2 0.2 1.2 0.2 1.2 4.0 Nulls 15.1 2.2 6.0 2.7 3.9 0.69.6 56.3 0.4 0.4 0.1 0.2 0.7 0.2 1.1 3.2

Thirty plants of each transgenic line were grown in a random arrangementin the greenhouse with parental control plants. T2 seeds were analysedfor oil content and exhibited an increase of about 2% in the oil content(total fatty acid level) compared to the total fatty acid content ofparental seeds (FIG. 8).

Expression of MGAT1 in Stably Transformed Trifolium repens Plants

A chimeric gene encoding M. musculus MGAT1 was used to transformTrifolium repens, another dicotyledonous plant. Vectors containing thechimeric genes 35S:MGAT1 and 35S:DGAT1 were introduced into A.tumefaciens via a standard electroporation procedure. Both vectors alsocontain a 35S:BAR selectable marker gene. The transformed Agrobacteriumcells were grown on solid LB media supplemented with kanamycin (50 mg/L)and rifampicin (25 mg/L) and incubated at 28° C. for two days. A singlecolony was used to initiate a fresh culture for each construct.Following 48 hours vigorous culture, the Agrobacterium cultures wereused to treat T. repens (cv. Haifa) cotyledons that had been dissectedfrom imbibed seed as described by Larkin et al. (1996). Followingco-cultivation for three days the explants were exposed to 5 mg/L PPT toselect transformed shoots and then transferred to rooting medium to formroots, before transfer to soil. A transformed plant containing MGAT1 wasobtained. The 35S promoter is expressed constitutively in cells of thetransformed plants. The oil content is increased in at least thevegetative tissues such as leaves.

Expression of MGAT in Stably Transformed Hordeum vulgare

A chimeric vector including M. musculus MGAT1 was used to produce stablytransformed Hordeum vulgare, a monocotyledonous plant. Vectorscontaining the chimeric genes Ubi:MGAT1 and Ubi:DGAT1 were constructedby cloning the entire M. musculus MGAT1 and A. thaliana DGAT1 codingregions separately into pWVEC8-Ubi. Vectors containing the chimericgenes Ubi:MGAT1 and Ubi:DGAT1 were introduced into A. tumefaciens strainAGL1 via a standard electroporation procedure. Transformed Agrobacteriumcells were grown on solid LB media supplemented with kanamycin (50 mg/L)and rifampicin (25 mg/L) and the plates incubated at 28° C. for twodays. A single colony of each was used to initiate fresh cultures.

Following 48 hours vigorous culture, the Agrobacterium cultures wereused to transform cells in immature embryos of barley (cv. GoldenPromise) according to published methods (Tingay et al., 1997; Bartlettet al., 2008) with some modifications. Briefly, embryos between 1.5 and2.5 mm in length were isolated from immature caryopses and the embryonicaxes removed. The resulting explants were co-cultivated for 2-3 dayswith the transgenic Agrobacterium and then cultured in the dark for 4-6weeks on media containing timentin and hygromycin to generateembryogenic callus before being moved to transition media in low lightconditions for two weeks. Callus was then transferred to regenerationmedia to allow for the regeneration of shoots and roots before transferto soil. Transformed plants were obtained and transferred to thegreenhouse. The MGAT1 coding region was expressed constitutively underthe control of the Ubi promoter in cells of the transformed plants.Transgenic plants were generated and their tissues analysed for oilcontent. Due to the low number of transgenic events obtained in a firsttransformation, no statistically significant conclusion could be drawnfrom the data.

The coding region of the mouse MGAT2 gene, codon optimised forexpression in plant cells, is substituted for the MGAT1 in theconstructs mentioned above, and introduced into Hordeum as describedabove. Vegetative tissues from the resultant transgenic plants areincreased for oil content.

Expression of MGAT in Yeast Cells

A chimeric vector including M. musculus MGAT1 was used to transformyeast, in this example Saccharomyces cerevisiae, a fungal microbesuitable for production of oil by fermentation. A genetic constructGal1:MGAT1 was made by inserting the entire coding region of a constructdesignated 0954364_MGAT_pMA, contained within an EcoRI fragment, intopYES2 at the EcoRI site, generating pJP3301. Similarly, a geneticconstruct Gal1:DGAT1, used here as a comparison and separately encodingthe enzyme A. thaliana DGAT1 was made by inserting the entire A.thaliana DGAT1 coding region into pYES2. These chimeric vectors wereintroduced into S. cerevisiae strain S288C by heat shock andtransformants were selected on yeast minimal medium (YMM) platescontaining 2% raffinose as the sole carbon source. Clonal inoculumcultures were established in liquid YMM with 2% raffinose as the solecarbon source. Experimental cultures were inoculated from these in YMMmedium containing 1% NP-40, to an initial OD600 of about 0.3. Cultureswere grown at 28° C. with shaking (about 100 rpm) until OD600 wasapproximately 1.0. At this point, galactose was added to a finalconcentration of 2% (w/v). Cultures were incubated at 25° C. withshaking for a further 48 hours prior to harvesting by centrifugation.Cell pellets were washed with water before being freeze-dried for lipidclass fractionation and quantification analysis as described inExample 1. The Gal promoter is expressed inducibly in the transformedyeast cells, increasing the oil content in the cells.

The coding region of the mouse MGAT2 gene, codon optimised forexpression in yeast cells, is substituted for the MGAT1 in theconstructs mentioned above, and introduced into yeast. The resultanttransgenic cells are increased for oil content. The genes are alsointroduced into the oleaginous yeast, Yarrowia lipolytica, to increaseoil content.

Expression of MGAT in Algal Cells Chlamydomonas reinhardtii

A chimeric vector including M. musculus MGAT1 is used to stablytransform algal cells. The genetic constructs designated 35S:MGAT1 ismade by cloning the MGAT1 coding region into a cloning vector containinga Cauliflower mosaic virus 35S promoter cassette and aparamomycin-resistance gene (aminoglycoside-O-phosphotransferase VIII)expressed by a C. reinhardtii RBCS2 promoter. 35S:MGAT1 is introducedseparately into a logarithmic culture of 5×10⁷ cc503, acell-wall-deficient strain of Chlamydomonas reinhardtii by a modifiedglass bead method (Kindle, 1990). Both vectors also contain the BLEresistance gene as a selectable marker gene. Briefly, a colony ofnon-transformed cells on a TAP agar plate kept at about 24° C. is grownto about 5×10⁶ cells/mL over four days, the resultant cells are pelletedat 3000 g for 3 minutes at room temperature and resuspended to produce5×10⁷ cells in 300 uL of TAP media. 300 uL of 0.6 mm diameter glassbeads, 0.6 μg plasmid in 5 μL and 100 μL of 20% PEG MW8000 are added andthe mix is vortexed at maximum speed for 30 seconds, then transferred to10 mL of TAP and incubated for 16 hours with shaking in the dark. Thecells are pelleted, resuspended in 200 μL of TAP then plated on TAPplates containing 5 mg/L zeocin and incubated in the dark for 3 weeks.Transformed colonies are subcultured to a fresh TAP+zeocin 5 mg/L plateafter which they are grown up under standard media conditions withzeocin selection. After harvesting by centrifugation, the cell pelletsare washed with water before being freeze-dried for lipid classfractionation and quantification analysis as described in Example 1. The35S:MGAT1 promoter is expressed constitutively in the transformed algalcells. The oil content of the cells is significantly increased.

The coding region of the mouse MGAT2 gene, codon optimised forexpression in plant cells, is substituted for the MGAT1 in the constructmentioned above, and introduced into Chlamydomonas. Oil content in theresultant transgenic cells is significantly increased.

Expression of MGAT in Stably Transformed Lupinus angustifolius

A chimeric vector including M. musculus MGAT1 is used to transformLupinus angustifolius, a leguminous plant. Chimeric vectors 35S:MGAT1and 35S: DGAT1 in Agrobacterium are used to transform L. angustifoliusas described by Pigeaire et al. (1997). Briefly, shoot apex explants areco-cultivated with transgenic Agrobacterium before being thoroughlywetted with PPT solution (2 mg/ml) and transferred onto a PPT-freeregeneration medium. The multiple axillary shoots developing from theshoot apices are excised onto a medium containing 20 mg/L PPT and thesurviving shoots transferred onto fresh medium containing 20 mg/L PPT.Healthy shoots are then transferred to soil. The 35S promoter isexpressed constitutively in cells of the transformed plants, increasingthe oil content in the vegetative tissues and the seeds. A seed specificpromoter is used to further increase the oil content in transgenicLupinus seeds.

The coding region of the mouse MGAT2 gene, codon optimised forexpression in plant cells, is substituted for the MGAT1 in theconstructs mentioned above, and introduced into Lupinus. Seeds andvegetative tissues from the resultant transgenic plants are increasedfor oil content.

Expression of MGAT in Stably Transformed Cells of Sorghum bicolor

A chimeric vector including M. musculus MGAT1 is used to stablytransform Sorghum bicolor. Ubi:MGAT1 and Ubi:DGAT1 in A. tumefaciensstrain AGL1 are used to transform Sorghum bicolor as described by Gurelet al. (2009). The Agrobacterium is first centrifuged at 5,000 rpm at 4°C. for 5 minutes and diluted to OD550=0.4 with liquid co-culture medium.Previously isolated immature embryos are then covered completely withthe Agrobacterium suspension for 15 minutes and then cultured, scutellumside up, on co-cultivation medium in the dark for 2 days at 24° C. Theimmature embryos are then transferred to callus-induction medium (CIM)with 100 mg/L carbenicillin to inhibit the growth of the Agrobacteriumand left for 4 weeks. Tissues are then transferred to regenerationmedium to shoot and root. The Ubi promoter is expressed constitutivelyin cells of the transformed plants, increasing the oil content in atleast the vegetative tissues.

The coding region of the mouse MGAT2 gene, codon optimised forexpression in plant cells, is substituted for the MGAT1 in theconstructs mentioned above, and introduced into Sorghum. Vegetativetissues from the resultant transgenic plants are increased for oilcontent.

Expression of MGAT in Stably Transformed Plants of Glycine max

A chimeric gene encoding M. musculus MGAT1 is used to stably transformGlycine max, another legume which may be used for oil production.35S:MGAT1 in Agrobacterium is used to transform G. max as described byZhang et al. (1999). The Agrobacterium is co-cultivated for three dayswith cotyledonary explants derived from five day old seedlings. Explantsare then cultured on Gamborg's B5 medium supplemented with 1.67 mg/L BAPand 5.0 mg/L glufosinate for four weeks after which explants aresubcultured to medium containing MS major and minor salts and B5vitamins (MS/B5) supplemented with 1.0 mg/L zeatin-riboside, 0.5 mg/LGA3 and 0.1 mg/L IAA amended with 1.7 mg/L or 2.0 mg/L glufosinate.Elongated shoots are rooted on a MS/B5 rooting medium supplemented with0.5 mg/L NAA without further glufosinate selection. The 35S promoter isexpressed constitutively in cells of the transformed plants, increasingthe oil content in the vegetative tissues and the seeds.

The coding region of the mouse MGAT2 gene, codon optimised forexpression in plant cells, is substituted for the MGAT1 in theconstructs mentioned above, and introduced into Glycine. Vegetativetissues and seeds from the resultant transgenic plants are increased foroil content.

Expression of MGAT in Stably Transformed Zea mays

A chimeric gene encoding M. musculus MGAT1 is used to stably transformZea mays. The vectors comprising 35S:MGAT1 and 35S:DGAT1 are used totransform Zea mays as described by Gould et al. (1991). Briefly, shootapex explants are co-cultivated with transgenic Agrobacterium for twodays before being transferred onto a MS salt media containing kanamycinand carbenicillin. After several rounds of sub-culture, transformedshoots and roots spontaneously form and are transplanted to soil. The35S promoter is expressed in cells of the transformed plants, increasingthe oil content in the vegetative tissues and the seeds.

The coding region of the mouse MGAT2 gene, codon optimised forexpression in plant cells, is substituted for the MGAT1 in theconstructs mentioned above, and introduced into Zea mays. Vegetativetissues and seeds from the resultant transgenic plants are increased foroil content. Alternatively, the MGAT coding regions are expressed underthe control of an endosperm specific promoter such as the zein promoter,or an embryo specific promoter obtained from a monocotyledonous plant,for increased expression and increased oil content in the seeds. Afurther chimeric gene encoding a GPAT with phosphatase activity, such asA. thaliana GPAT4 or GPAT6 is introduced into Zea mays in combinationwith the MGAT, further increasing the oil content in corn seeds.

Expression of MGAT in Stably Transformed Elaeis guineensis (Palm Oil)

A chimeric gene encoding M. musculus MGAT1 is used to stably transformElaeis guineensis. Chimeric vectors designated Ubi:MGAT1 and Ubi:DGAT1in Agrobacterium are used. Following 48 hours vigorous culture, thecells are used to transform Elaeis guineensis as described by Izawati etal. (2009). The Ubi promoter is expressed constitutively in cells of thetransformed plants, increasing the oil content in at least the fruitsand seeds, and may be used to obtain oil.

The coding region of the mouse MGAT2 gene, codon optimised forexpression in plant cells, is substituted for the MGAT1 in theconstructs mentioned above, and introduced into Elaeis. Seeds from theresultant transgenic plants are increased for oil content.

Expression of MGAT in Stably Transformed Avena sativa (Oats)

A chimeric gene encoding M. musculus MGAT1 is used to stably transformAvena sativa, another monocotyledonous plant. Chimeric vectorsdesignated Ubi: MGAT1 and Ubi: DGAT1, as described above and bothcontaining a Ubi:BAR selectable marker, are used to transform Avenasativa as described by Zhang et al. (1999).

The coding region of the mouse MGAT2 gene, codon optimised forexpression in plant cells, is substituted for the MGAT1 in theconstructs mentioned above, and introduced into Avena. Seeds from theresultant transgenic plants are increased for oil content.

Example 7 Engineering a MGAT with DGAT Activity

An MGAT with altered DGAT activity, especially increased DGAT activityand potentially increased MGAT activity may be engineered by performingrandom mutagenesis, targeted mutagenesis, or saturation mutagenesis onMGAT gene(s) of interest or by subjecting different MGAT and/or DGATgenes to DNA shuffling. DGAT function can be positively screened for byusing, for example, a yeast strain that has an absolute requirement forTAG-synthesis complementation when fed free fatty acids, such as strainH1246 which contains mutations in four genes (DGA1, LRO1, ARE1, ARE2).Transforming the MGAT variants in such a strain and then supplying thetransformed yeast with a concentration of free fatty acids that preventscomplementation by the wildtype MGAT gene will only allow the growth ofvariants with increased TAG-synthesis capability due to improved DGATactivity. The MGAT activity of these mutated genes can be determined byfeeding labelled sn-1 or sn-2 MAG and quantifying the production oflabelled DAG. Several rounds of directed evolution in combination withrational protein design would result in the production of a novel MGATgene with similar MGAT and DGAT activities.

The gene coding for the M. musculus MGAT1 acyltransferase was subjectedto error prone PCR using Taq DNA polymerase in the presence of 0.15 mMMnCl₂ to introduce random mutations. The randomized coding regions werethen used as megaprimers to amplify the entire yeast expression vectorusing high fidelity PCR reaction conditions. Sequencing of 9099 bp ofrecovered, mutagenised DNA revealed a mutational frequency of about0.8%, corresponding to 8 mutations per gene or, on average, 5.3 aminoacid substitutions per polypeptide. The entire mutagenised library wastransformed into E. coli DH5a for storage at −80° C. and plasmidpreparation. The size of the MGAT1 library was estimated at 3.8356E6clones. A copy of the MGAT1 library was transformed into the yeaststrain H1246, resulting in a library size of 3E6 clones. The MGAT1library as well as a pYES2 negative control, transformed into S.cerevisiae H1246, were subjected to 8 selection rounds, each consistingof (re)diluting cultures in minimal induction medium (1% raffinose+2%galactose; diluted to OD₆₀₀=0.35-0.7) in the presence of C18:1 freefatty acid at a 1 mM final concentration. Negative controls consisted ofidentical cultures grown simultaneously in minimal medium containingglucose (2%) and in the absence of C18:1 free fatty acid. After 8selection rounds, an aliquot of the selected MGAT1 library was plated onminimal medium containing glucose (2%). A total of 120 colonies weregrown in 240 μl minimal induction medium in 96 microtiter plates andassayed for neutral lipid yield using a Nile Red fluorescence assay asdescribed by Siloto et al. (2009). Plasmid minipreps were prepared from113 clones (=top 6%) that displayed the highest TAG levels.

The entire MGAT1 coding region of the selected clones is sequenced toidentify the number of unique mutants and to identify the nature of theselected mutations. Unique MGAT1 mutants are retransformed into S.cerevisiae H1246 for in vitro MGAT and DGAT assays using labelled MAGand C18:1 substrates respectively (see Example 5). Selected MGAT1variants are found to exhibit increased DGAT activity compared to thewild type acyltransferase, whilst MGAT activity is possibly increased aswell.

MGAT1 variants displaying increased MGAT and/or DGAT activities are usedas parents in a DNA shuffling reaction. The resulting library issubjected to a similar selection system as described above resulting infurther improvement of general acyltransferase activity. In addition,free fatty acids other than C18:1 are added to the growth medium toselect for MGAT1 variants displaying altered acyl-donor specificities.

Example 8 Constitutive Expression of the A. thaliana DiacylglycerolAcyltransferase 2 in Plants

Expression of the A. thaliana DGAT2 in yeast (Weselake et al., 2009) andinsect cells (Lardizabal et al., 2001) did not demonstrate DGATactivity. Similarly, the DGAT2 was not able to complement an A. thalianaDGAT1 knockout (Weselake et al., 2009). The enzyme activity of the A.thaliana DGAT2 in leaf tissue was determined using a N. benthamianatransient expression system as described in Example 1. The A. thalianaDGAT2 (accession Q9ASU1) was obtained by genomic PCR and cloned into abinary expression vector under the control of the 35S promoter togenerate 35S:DGAT2. This chimeric vector was introduced into A.tumefaciens strain AGL1 and cells from cultures of these infiltratedinto leaf tissue of N. benthamiana plants in a 24° C. growth room using35S:DGAT1 as a control. Several direct comparisons were infiltrated withthe samples being compared located on either side of the same leafExperiments were performed in triplicate. Following infiltration theplants were grown for a further five days before leaf discs were takenand freeze-dried for lipid class fractionation and quantificationanalysis as described in Example 1. This analysis revealed that bothDGAT1 and DGAT2 were functioning to increase leaf oil levels inNicotiana benthamiana (Table 8).

Leaf tissue transformed with the 35S:p19 construct (negative control)contained an average of 25 μg TAG/100 mg dry leaf weight. Leaf tissuetransformed with the 35S:p19 and 35S:DGAT1 constructs (positive control)contained an average of 241 μg TAG/100 mg dry leaf weight. Leaf tissuetransformed with the 35S:p19 and 35S:DGAT2 constructs contained anaverage of 551 μg TAG/100 mg dry leaf weight.

The data described above demonstrates that the A. thaliana DGAT2 enzymeis more active than the A. thaliana DGAT1 enzyme in promoting TAGaccumulation in leaf tissue. Expression of the DGAT2 gene resulted in229% as much TAG accumulation in leaf tissue compared to when the TAGamount from DGAT1 over-expressed was set as relative 100% (FIG. 9).

Transiently-transformed N. benthamiana leaf tissues expressing P19 alone(control), or P19 with either AtDGAT1 or AtDGAT2 were also used toprepare microsomes for in vitro assays of enzyme activity. A DGATbiochemical assay was performed using microsomes corresponding to 50 μgprotein and adding 10 nmole [14]C6:0-DAG and 5 nmole acyl-CoA, in 50 mMHepes buffer, pH 7.2, containing 5 mM MgCl₂, and 1% BSA in a finalvolume of 100 μL for each assay. The assays were conducted at 30° C. for30 minutes. Total lipid from each assay was extracted and samples loadedon TLC plates, which were developed using a hexane:DEE:Hac solvent(70:30:1 vol:vol:vol). The amount of radioactivity in DAG and TAG spotswas quantified by PhosphorImage measurement. The percentage of DAGconverted to TAG was calculated for each of the microsome preparations.

Some endogenous DGAT activity was detected in the N. benthamiana leaves,as the P19 control assay showed low levels of TAG production. Theexpression of AtDGAT1 yielded increased DGAT activity relative to theP19 control when the assays were supplemented with either C18:1-CoA orC18:2-CoA, but not when supplemented with C18:3-CoA, where the levels ofTAG for the P19 control and the AtDGAT1 were similar. However, in all ofthe microsomal assays when AtDGAT2 was expressed in the leaf tissues,greater levels of DGAT activity (TAG production) were observed comparedto the AtDGAT1 microsomes. Greater levels of TAG production wereobserved when the microsomes were supplemented with either C18:2-CoA orC18:3-CoA relative to C18:1-CoA (FIG. 10). This indicated that DGAT2 hada different substrate preference, in particular for C18:3-CoA (ALA),than DGAT1.

Example 9 Co-Expression of MGAT and GPAT in Transgenic Seed

Yang et al. (2010) described two glycerol-3-phosphate acyltransferases(GPAT4 and GPAT6) from A. thaliana both having a sn-2 preference (i.e.preferentially forming sn-2 MAG rather than sn-1/3 MAG) and phosphataseactivity, which were able to produce sn-2 MAG from G-3-P (FIG. 1). Theseenzymes were proposed to be part of the cutin synthesis pathway. GPAT4and GPAT6 were not expressed highly in seed tissue. Combining such abifunctional GPAT/phosphatase with a MGAT yields a novel DAG synthesispathway using G-3-P as a substrate that can replace or supplement thetypical Kennedy Pathway for DAG synthesis in plants, particularly inoilseeds, or other cells, which results in increased oil content, inparticular TAG levels.

Chimeric DNAs designated pJP3382 and pJP3383, encoding the A. thalianaGPAT4 and GPAT6, respectively, together with the M. musculus MGAT2 forexpression in plant seeds were made by first inserting the entire MGAT2coding region, contained within a SwaI fragment, into pJP3362 at theSmaI site to yield pJP3378. pJP3362 was a binary expression vectorcontaining empty FAE1 and FP1 expression cassettes and a kanamycinresistance gene as a selectable marker. The A. thaliana GPAT4 wasamplified from cDNA and cloned into pJP3378 at the NotI site to yieldpJP3382 in which the GPAT4 was expressed by the truncated napinpromoter, FP1, and the MGAT2 was expressed by the A. thaliana FAE1promoter. Similarly, the A. thaliana GPAT6 was amplified from cDNA andcloned into pJP3378 at the NotI site to yield pJP3384 in which the GPAT6was operably linked to the truncated napin promoter, FP1, and the MGAT2was expressed by the A. thaliana FAE1 promoter. pJP3382 and pJP3383 weretransformed into A. thaliana (ecotype Columbia) by the floral dipmethod. Seeds from the treated plants were plated onto media containingthe antibiotic, kanamycin, to select for progeny plants (T1 plants)which were transformed. Transgenic seedlings were transferred to soiland grown in the greenhouse. Expression of the transgenes in thedeveloping embryos was determined. Transgenic plants with the highestlevel of expression and which show a 3:1 ratio fortransgenic:non-transgenic plants per line, indicative of a single locusof insertion of the transgenes, are selected and grown to maturity.Seeds were obtained from these plants (T2) which included some whichwere homozygous for the transgenes. 30 to 32 (T2 plants) from each linewere grown in pots of soil in a random arrangement in the greenhousewith control plants, and the lipid content, TAG content and fatty acidcompositions of the resultant seed was determined. The total fatty acidcontent (as determined from the total FAME), in particular the TAGcontent of the seeds comprising both a MGAT and a GPAT4 or GPAT6 wassubstantially and significantly increased by nearly 3% (absolute level)or by about 9% (relative increase) over the controls, and increasedrelative to seeds comprising the MGAT alone or the A. thaliana DGAT1alone (FIG. 11).

The coding region of the mouse MGAT2 gene, codon optimised forexpression in plant cells, was introduced into Brassica napus togetherwith a chimeric gene encoding Arabidopsis GPAT4. Seeds from theresultant transgenic plants were harvested and some were analysed. Datafrom these preliminary analyses showed variability in the oil contentand fatty acid composition, probably due to the plants being grown atdifferent times and under different environmental conditions. Seeds areplanted to produce progeny plants, and progeny seeds are harvested.

Example 10 Testing the Effect of GPAT4 and GPAT6 on MGAT-Mediated TAGIncrease by GPAT Silencing and Mutation

The GPAT family is large and all known members contain two conserveddomains, a plsC acyltransferase domain and a HAD-like hydrolasesuperfamily domain. In addition to this, A. thaliana GPAT4-8 all containan N-terminal region homologous to a phosphoserine phosphatase domain.A. thaliana GPAT4 and GPAT6 both contain conserved residues that areknown to be critical to phosphatase activity (Yang et al., 2010).

Degenerate primers based on the conserved amino acid sequenceGDLVICPEGTTCREP (SEQ ID NO:228) were designed to amplify fragments on N.benthamiana GPATs expressed in leaf tissue. 3′ RACE will be performedusing these primers and oligo-dT reverse primers on RNA isolated from N.benthamiana leaf tissue. GPATs with phosphatase activity (i.e.GPAT4/6-like) will be identified by their homology with the N-terminalphosphoserine phosphatase domain region described above. 35S-driven RNAiconstructs targeting these genes will be generated and transformed in A.tumefaciens strain AGL1. Similarly, a 35S:V2 construct containing the V2viral silencing-suppressor protein will be transformed in A. tumefaciensstrain AGL1. V2 is known to suppress the native plant silencingmechanism to allow effective transient expression but also allowRNAi-based gene silencing to function.

TAG accumulation will then be compared between transiently-transformedleaf samples infiltrated with the following strain mixtures: 1) 35S:V2(negative control); 2) 35S:V2+35S:MGAT2 (positive control); 3)35S:V2+GPAT−RNAi; 4) 35S:V2+GPAT−RNAi+35S:MGAT2. It is expected that the35S:V2+GPAT−RNAi+35S:MGAT2 mixture will result in less TAG accumulationthan the 35S:V2+35S:MGAT2 sample due to interrupted sn-2 MAG synthesisresulting from the GPAT silencing.

A similar experiment will be performed using A. thaliana and N.benthamiana GPAT4/6-like sequences which are mutated to remove theconserved residues that are known to be critical to phosphatase activity(Yang et al., 2010). These mutated genes (known collectively asGPAT4/6-delta) will then be cloned into 35S-driven expression binaryvectors and transformed in A. tumefaciens. TAG accumulation will then becompared between transiently-transformed leaf samples infiltrated withthe following strain mixtures: 1) 35S:p19 (negative control); 2)35S:p19+35S:MGAT2 (positive control); 3) 35S:p19+GPAT4/6-delta; 4)35S:p19+GPAT4/6-delta+35S:MGAT2. It is expected that the35S:p19+GPAT4/6-delta+35S:MGAT2 mixture will result in less TAGaccumulation than the 35S:p19+35S:MGAT2 sample due to interrupted sn-2MAG synthesis resulting from the GPAT mutation. Whilst the native N.benthamiana GPAT4/6-like genes will be present in this experiment it isexpected that high-level expression of the GPAT4/6-delta constructs willoutcompete the endogenous genes for access to the G-3-P substrate.

Example 11 Constitutive Expression of a Diacylglycerol Acyltransferaseand WRI1 Transcription Factor in Plant Cells

A vector designated 35S-pORE04 was made by inserting a PstI fragmentcontaining a 35S promoter into the SfoI site of vector pORE04 after T4DNA polymerase treatment to blunt the ends (Coutu et al., 2007). Agenetic construct 35S:Arath-DGAT1 encoding the A. thalianadiacylglycerol acyltransferase DGAT1 (Bouvier-Nave et al., 2000) wasmade. Example 3 of WO 2009/129582 describes the construction of AtDGAT1in pXZP163. A PCR amplified fragment with KpnI and EcoRV ends was madefrom pXZP163 and inserted into pENTR11 to generate pXZP513E. The entireAtDGAT1 coding region of pXZP513E contained within a BamHI-EcoRVfragment was inserted into 35S-pORE04 at the BamHI-EcoRV site,generating pJP2078. A synthetic fragment, Arath-WRI1, coding for the A.thaliana WRI1 transcription factor (Cernac and Benning, 2004), flankedby EcoRI restriction sites and codon optimized for B. napus, wassynthesized. A genetic construct designated 35S:Arath-WRI1 was made bycloning the entire coding region of Arath-WRI1, flanked by EcoRI sitesinto 35S-pORE04 at the EcoRI site generating pJP3414. Expression of thegenes in N. benthamiana leaf tissue was performed according to thetransient expression system as described in Example 1.

Quantification of TAG levels of infiltrated N. benthamiana leaves byIatroscan revealed that the combined expression of the A. thaliana DGAT1and WRI1 genes resulted in 4.5-fold and 14.3-fold increased TAG contentcompared to expression of WRI1 and the V2 negative control respectively(Table 9). This corresponded to an average and maximum observed TAGyield per leaf dry weight of 5.7% and 6.51% respectively (Table 9 andFIG. 12). The increase in leaf oil was not solely due to the activity ofthe overexpressed DGAT1 acyltransferase as was apparent in the reducedTAG levels when WRI1 was left out of the combination. Furthermore, asynergistic effect was observed accounting for 48% of the total TAGincrease.

Both DGAT1 and WRI1 constructs also led to increased oleic acid levelsat the expense of linoleic acid in TAG fractions of infiltrated N.benthamiana leaves (Table 10). These results confirm recent findings byAndrianov et al. (2010) who reported similar shifts in the TAG,phospholipid and TFA lipid fractions of transgenic tobacco plantstransformed with the A. thaliana DGAT1 acyltransferase. However, whenDGAT1 and WRI1 genes were co-expressed, a synergistic effect wasobserved on the accumulation of oleic acid in the N. benthamianaleaves—this synergism accounted for an estimated at 52% of the totaloleic acid content when both genes were expressed. The unexpectedsynergistic effects on both TAG accumulation and oleic acid levels intransgenic N. benthamiana leaves demonstrated the potential ofsimultaneously up-regulating fatty acid biosynthesis and acyl uptakeinto non-polar lipid such as TAG in vegetative tissues, two metabolicprocesses that are highly active in developing oilseeds.

The transient expression experiment was repeated except that the P19viral silencing suppressor as substituted for the V2 suppressor, andwith careful comparison of samples on the same leaf to avoid anyleaf-to-leaf variation. For this, a chimeric 35S:P19 construct forexpression of the tomato bushy stunt virus P19 viral silencingsuppressor protein (Wood et al., 2009) was separately introduced into A.tumefaciens GV3101 for co-infiltration.

Quantification of TAG levels of infiltrated N. benthamiana leaves byIatroscan in this experiment revealed that the combined transientexpression of the A. thaliana DGAT1 and WRI1 genes resulted in 141-foldincreased TAG content compared to P19 negative control (FIG. 13). Whencompared to the expression of the DGAT1 and WRI1 genes separately on thesame leaf, the combined infiltration increased TAG levels by 17- and5-fold respectively. Once again, the co-expression of both genes had asynergistic (larger than additive) effect on leaf oil accumulation withthe synergistic component accounting for 73% of the total TAG increase.The greater extent of the increased TAG content in this experiment(141-fold) compared to the previous experiment (14.3-fold) may have beendue to use of the P19 silencing suppressor rather than V2 and thereforeincreased gene expression from the transgenes.

Table 11 shows the fatty acid composition of the TAG. When DGAT1 andWRI1 genes were co-expressed in N. benthamiana, a synergistic effect wasonce again observed on the level of oleic acid accumulation in the leafTAG fraction. This increase was largely at the expense of the mediumchain unsaturated fatty acids palmitic acid and stearic acid (Table 11).Linoleic acid was also increased which can be explained by the higheroleic acid substrate levels available to the endogenous FAD2Δ12-desaturase. Individual expression of the DGAT1 and WRI1 genes in N.benthamiana led to intermediate changes in the TAG profile without asgreat an increase in oleic acid. In addition, but in contrast to thefirst experiment, higher levels of α-linolenic acid (ALA) were detectedwhile this was observed to a lesser extent upon the DGAT1 and WRI1co-expression in leaf tissue.

The observed synergistic effect of DGAT1 and WRI1 expression on TAGbiosynthesis was confirmed in more detail by comparing the effect ofintroduction into N. benthamiana of both genes individually or incombination, compared to introduction of a P19 gene alone as a control,within the same leaf. This was beneficial in reducing leaf to leafvariation. In addition, the number of replicates was increased to 5 andsamples were pooled across different leaves from the same plant toimprove the quality of the data. Results are presented in Table 12.

TABLE 8 Fatty acid profile and quantification of TAG in triplicateNicotiana benthamiana leaf tissue transiently transformed with the35S:p19, 35S:DGAT1 and 35S:DGAT2 constructs. Sample C16:0 16:1w13tC16:1d7 16:3w3 C18:0 C18:1 C18:1d11 C18:2 C18:3 P19 44.7 0.1 0.0 0.033.9 1.2 0.0 6.5 12.7 44.1 1.7 0.0 0.0 15.3 2.0 0.0 15.2 19.5 43.3 1.50.0 0.0 10.5 1.5 0.0 17.2 23.9 P19 + AtDGAT1 36.3 0.5 0.1 0.4 11.6 2.30.3 17.8 24.5 33.6 0.5 0.1 0.4 11.2 2.9 0.3 23.1 21.5 36.8 0.5 0.0 0.012.4 2.9 0.4 21.3 19.3 P19 + AtDGAT2 18.6 0.3 0.1 0.5 9.3 7.7 0.4 28.033.1 17.5 0.3 0.1 0.3 10.2 9.9 0.5 32.7 26.5 18.4 0.3 0.1 0.3 9.8 7.50.5 32.3 29.1 Sample C20:0 20:1d11 20:2 20:3n3 C22:0 C24:0 μg/100 mg DWP19 0.9 0.0 0.0 0.0 0.0 0.0 43.29 2.2 0.0 0.0 0.0 0.0 0.0 23.12 2.2 0.00.0 0.0 0.0 0.0 38.35 P19 + AtDGAT1 3.6 0.0 0.0 0.2 1.5 0.2 144.77 3.80.0 0.0 0.2 1.5 0.9 145.34 3.9 0.0 0.0 0.0 1.5 1.0 90.04 P19 + AtDGAT21.1 0.2 0.1 0.1 0.2 0.3 439.25 1.2 0.1 0.0 0.1 0.2 0.4 282.50 1.2 0.00.0 0.0 0.3 0.2 208.40

TABLE 9 TAG levels in triplicate Nicotiana benthamiana leaf tissuetransiently transformed with 35S:V2, 35S:DGAT1 and 35S:WRI1. TAG TAGControl (% dry Genes (% dry combination weight) ¹ expressed weight) ¹Ratio ² V2 0.41 ± 0.10 V2, WRI1, 5.71 ± 0.63 14.28 ± 1.89  DGAT1 V2,WRI1 1.16 ± 0.60 V2, WRI1, 4.25 ± 0.64 4.45 ± 2.24 DGAT1 V2, DGAT1 1.52± 0.34 V2, WRI1, 4.76 ± 0.50 3.22 ± 0.75 DGAT1 ¹ Average of threedifferent infiltrated leaves as quantified by Iatroscan ² Average ratiobased on side-by-side comparisons on the same leaves

TABLE 10 Fatty acid composition of TAG produced in Nicotiana benthamianaleaf tissue transiently transformed with 35S:V2, 35S:DGAT1 and 35S:WRI1(data from triplicate infiltrations). V2, WRI1, V2, WRI1, Fatty acid V2V2, WRI1, DGAT1 V2, WRI1 DGAT1 V2, DGAT1 DGAT1 C14:0 0 0 0 0 0 0C14:1^(Δ9)  1.26 ± 2.18 0.05 ± 0.10 0 0.04 ± 0.08 0 0.04 ± 0.07 C16:046.12 ± 0.97 30.60 ± 0.41  50.09 ± 6.27  31.32 ± 3.31  35.44 ± 0.80 26.61 ± 1.41  C16:1^(Δ9) 0 0.13 ± 0.11 0 0.07 ± 0.11 0 0.13 ± 0.12 C18:013.44 ± 1.65 9.93 ± 1.19 9.28 ± 0.81 9.93 ± 0.53 12.20 ± 1.03  8.76 ±0.91 C18:1^(Δ9)  5.09 ± 5.32 36.78 ± 2.23  9.72 ± 5.08 27.97 ± 4.19 8.77 ± 1.97 32.41 ± 1.39  C18:1^(Δ11) 0 0.56 ± 0.04 0 0.51 ± 0.04 0 0.55± 0.04 C18:2^(Δ9,12) 14.12 ± 0.75 11.83 ± 0.75  13.26 ± 1.95  16.45 ±3.88  18.93 ± 0.77  17.03 ± 1.36  C18:3^(Δ9,12,15) 19.98 ± 6.33 4.77 ±1.17 17.10 ± 4.31  8.75 ± 2.13 16.12 ± 3.36  9.57 ± 0.61 C20:0 0 2.63 ±0.27 0.54 ± 0.93 2.53 ± 0.16 4.25 ± 0.33 2.43 ± 0.26 C22:0 0 1.56 ± 0.1 0 1.38 ± 0.03 2.37 ± 0.11 1.40 ± 0.13 C24:0 0 1.17 ± 0.15 0 1.05 ± 0.071.92 ± 0.16 1.07 ± 0.16

TABLE 11 Fatty acid composition of TAG produced in N. benthamiana leaftissues transiently transformed with 35S:P19 (control), 35S:WRI1 and/or35S:DGAT1 constructs. P19 + Fatty P19 + P19 + WRI1 + acid P19 WRI1 DGAT1DGAT1 C14:0 3.0 ± 2.2 0.6 ± 0.1 0.2 ± 0.1 0.1 ± 0.0 C16:0 46.5 ± 4.1 48.7 ± 11.5 28.4 ± 0.3  28.1 ± 1.0  C16:1^(Δ3t) 1.3 ± 2.2 0.3 ± 0.3 0.5± 0.0 0.3 ± 0.0 C16:1^(Δ9) 0.0 0.9 ± 0.2 0.2 ± 0.0 0.4 ± 0.1C16:3^(Δ7,12,15) 0.0 0.2 ± 0.2 0.5 ± 0.1 0.3 ± 0.0 C18:0 18.7 ± 4.7  7.9± 2.6 11.5 ± 0.6  7.2 ± 0.4 C18:1^(Δ9) 5.5 ± 1.3 3.9 ± 0.3 6.3 ± 0.219.4 ± 2.7  C18:1^(Δ11) 0.0 0.6 ± 0.1 0.2 ± 0.0 0.6 ± 0.1 C18:2^(Δ9,12)11.3 ± 4.2  12.7 ± 3.6  25.2 ± 0.5  26.3 ± 1.0  C18:3^(Δ9,12,15) 9.3 ±3.6 21.6 ± 10.3 18.1 ± 0.6  11.2 ± 0.7  C20:0 2.7 ± 0.2 1.4 ± 0.5 4.4 ±0.1 2.7 ± 0.1 C20:1^(Δ11) 0.0 0.0 0.3 ± 0.0 0.3 ± 0.0 C20:2^(Δ11,14) 0.00.0 0.1 ± 0.1 0.2 ± 0.0 C20:3^(Δ11,14,17) 0.0 0.0 0.1 ± 0.0 0.1 ± 0.0C22:0 1.5 ± 0.1 0.7 ± 0.1 2.3 ± 0.0 1.8 ± 0.1 C24:0 0.4 ± 0.6 0.5 ± 0.21.6 ± 0.1 1.0 ± 0.1

TABLE 12 Comparison of WRI1 + DGAT1 together with the single genes TAGlevel Ratio Gene (% dry (compared combination weight) to P19) P19(control) 0.01 ± 0.00 1 P19 + WRI1 0.08 ± 0.04 8 P19 + DGAT1 0.27 ± 0.0327 P19 + WRI1 + 1.29 ± 0.26 129 DGAT1

Based on the individual effects of both DGAT1 and WRI1 genes uponexpression in N. benthamiana, in the presence of merely an additiveeffect but the absence of any synergistic effect, the present inventorsexpected a TAG level of about 0.35 or a 35-fold increase compared to theP19 negative control. However, the introduction of both genes resultedin TAG levels that were 129-fold higher than the P19 control. Based onthese results, the present inventors estimated the additive effect andthe synergistic effect on TAG accumulation as 26.9% and 73.1%,respectively. In addition, when the fatty acid composition of the totallipid in the leaf samples was analysed by GC, a synergistic effect wasobserved on C18:1^(Δ9) levels in the TAG fraction of N. benthamianaleaves infiltrated with WRI1 and DGAT1 (3 repeats each). The data isshown in Table 11.

For seed-specific expression of the WRI1+DGAT1 combination, Arabidopsisthaliana was transformed with a binary vector construct including achimeric DNA having both pFAE1::WRI1 and pCln2::DGAT1 genes, or, forcomparison, the single genes pFAE1::WRI1 or pCln2::DGAT1. T1 seeds wereharvested from the plants. The oil content of the seeds is determined.The seeds have an increased oil content.

Example 12 Constitutive Expression of a Monoacylglycerol Acyltransferaseand WRI1 Transcription Factor in Plant Cells

A chimeric DNA encoding the Mus musculus MGAT2 (Cao et al., 2003; Yenand Farese, 2003) and codon-optimised for B. napus was synthesized byGeneart. A genetic construct designated 35S:Musmu-MGAT2 was made byinserting the entire coding region of 1022341_MusmuMGAT2, containedwithin an EcoRI fragment, into pJP3343 at the EcoRI site, generatingpJP3347. Cloning of the 35S:Arath-WRI1 construct is described in Example11. Transient expression in N. benthamiana leaf tissue was performed asdescribed in Example 1.

When the mouse MGAT2 and the A. thaliana WRI1 transcription vector werecoexpressed, average N. benthamiana leaf TAG levels were increased by3.3-fold compared to the expression of WRI1 alone (Table 13). Inaddition, the expression of the two genes resulted in a small (29%)synergistic effect on the accumulation of leaf TAG. The TAG levelobtained with the MGAT2 gene in the presence of WRI1 was 3.78% asquantified by Iatroscan (FIG. 12). The similar results obtained with theanimal MGAT2 and plant DGAT1 acyltransferases in combination with the A.thaliana WRI1 suggests that a synergistic effect might be a generalphenomenon when WRI and acyltransferases are overexpressed in non-oilaccumulating vegetative plant tissues.

The experiment was repeated to introduce constructs for expressingV2+MGAT2 compared to V2+MGAT2+WRI1, such that infiltrated leaf sampleswere pooled across three leaves from the same plant, for two plantseach. In total, each combination therefore had 6 replicateinfiltrations. This yielded a smaller standard deviation than poolingleaf samples between different plants as was done in the firstexperiments. The data from this experiment is shown in Table 14. Earlierresults (Table 13) were confirmed. Although absolute TAG levels aredifferent (inherent to the Benth assay and also different pooling ofsamples), relative increase in TAG when WRI1 is co-expressed withV2+MGAT2 are similar (2.45- and 2.65-fold).

Example 13 Constitutive Expression of a MonoacylglycerolAcyltransferase, Diacylglycerol Acyltransferase and WRI1 TranscriptionFactor in Plant Cells

The genes coding for the A. thaliana diacylglycerol acyltransferaseDGAT1, the mouse monoacylglycerol acyltransferase MGAT2 and the A.thaliana WRI1 were expressed in different combinations in N. benthamianaleaf tissue according to the transient expression system as described inExample 1. A detailed description of the different constructs can befound in Examples 11 and 12.

The combined expression of the DGAT1, WRI1 and MGAT2 genes resulted inan almost 3-fold further average TAG increase when compared to theexpression of the latter two (Table 15). The maximum observed TAG yieldobtained was 7.28% as quantified by Iatroscan (FIG. 12). Leaf TAG levelswere not significantly affected when the gene of the mouse MGAT2acyltransferase was left out this combination. Results described inExample 16, however, clearly demonstrated the positive effect of themouse MGAT2 on the biosynthesis of neutral lipids in N. benthamianaleaves when expressed in combination with WRI1, DGAT1 and the Sesamumindicum oleosin protein.

TABLE 13 TAG levels in triplicate Nicotiana benthamiana leaf tissuetransiently transformed with 35S:V2, 35S:MGAT2 and 35S:WRI1. TAG TAGControl (% dry Genes (% dry combination weight) ¹ expressed weight) ¹Ratio ² V2, WRI1 0.93 ± 0.37 V2, WRI1, 2.88 ± 0.56 3.30 ± 0.85 MGAT2 V2,MGAT2 1.56 ± 0.76 V2, WRI1, 3.15 ± 1.05 2.45 ± 1.73 MGAT2 ¹ Average ofthree different infiltrated leaves as quantified by Iatroscan ² Averageratio based on side-by-side comparisons on the same leaves

TABLE 14 TAG content of infiltrated N. benthamiana leaf samples. GeneTAG (% dry combination weight) Ratio V2 + MGAT2 0.34 ± 0.04 2.65 V2 +MGAT2 + WRI1  0.9 ± 0.19

TABLE 15 TAG levels in triplicate Nicotiana benthamiana leaf tissuetransiently transformed with 35S:V2, 35S:MGAT2, 35S:DGAT1 and 35S:WRI1.TAG TAG Control (% dry Genes (% dry combination weight) ¹ expressedweight) ¹ Ratio ² V2, WRI1, 3.35 ± 0.29 V2, WRI1, 3.15 ± 0.49 0.94 ±0.01 DGAT1 MGAT2, DGAT1 V2, WRI1, 1.72 ± 0.56 V2, WRI1, 4.62 ± 0.47 2.88± 0.90 MGAT2 MGAT2, DGAT1 ¹ Average of three different infiltratedleaves as quantified by Iatroscan ² Average ratio based on side-by-sidecomparisons on the same leaves

Additional data was obtained from a further experiment where leafsamples were pooling across leaves within same plant, 6 replicates ofeach. The data is shown in Table 16.

TABLE 16 TAG content of infiltrated N. benthamiana leaf samples. GeneTAG (% dry combination weight) Ratio V2 + MGAT2 + 1.08 ± 0.1  2.06 DGAT1V2 + MGAT2 + 2.22 ± 0.31 DGAT1 + WRI1

Example 14 Constitutive Expression of a MonoacylglycerolAcyltransferase, Diacycerol Acyltransferase, WRI1 Transcription Factorand glycerol-3-phosphate Acyltransferase in Plant Cells

A 35S:GPAT4 genetic construct was made by cloning the A. thaliana GPAT4gene (Zheng et al., 2003) from total RNA isolated from developingsiliques, followed by insertion as an EcoRI fragment into pJP3343resulting in pJP3344. Other constructs are described in Examples 11 and12. Transient expression in N. benthamiana leaf tissue was performed asdescribed in Example 1.

Transient expression of the A. thaliana GPAT4 acyltransferase incombination with MGAT2, DGAT1 and WRI1 led to a small decrease in the N.benthamiana leaf TAG content as quantified by Iatroscan (Table 17). TheTAG level (5.78%) was also found to be lower when GPAT4 was included inthe infiltration mixture (FIG. 12). However, this finding does not ruleout the hypothesis of sn2-MAG synthesis from G3P as catalysed by theGPAT4 acyltransferase. Rather, it suggests that this catalytic step isunlikely to be rate limiting in leaf tissue due to the high expressionlevels of the endogenous GPAT4 gene (Li et al., 2007). Moreover, the A.thaliana GPAT8 acyltransferase displays a similar expression profile asGPAT4 and has been shown to exhibit an overlapping function (Li et al.,2007). In developing seeds the expression levels of GPAT4 and GPAT8 arelow. As a result, coexpression of GPAT4 in a seed context might becrucial to ensure sufficient sn2-MAG substrate for a heterologousexpressed MGAT acyltransferase.

TABLE 17 TAG levels in triplicate Nicotiana benthamiana leaf tissuetransiently transformed with 35S:V2, 35S:MGAT2, 35S:DGAT1, 35S:WRI1 and35S:GPAT4. Control combination TAG (% dry weight)¹ Genes expressed TAG(% dry weight)¹ Ratio² V2, WRI1, MGAT2, 4.14 ± 0.82 V2, WRI1, MGAT2,3.11 ± 0.20 0.77 ± 0.13 DGAT1 DGAT1, GPAT4 V2, WRI1, DGAT1, 2.76 ± 0.74V2, WRI1, MGAT2, 4.05 ± 1.24 1.47 ± 0.22 GPAT4 DGAT1, GPAT4

Additional data was obtained from a further experiment where leafsamples were pooling across leaves within same plant, 6 replicates ofeach. The data is shown in Table 18.

TABLE 18 TAG content of infiltrated N. benthamiana leaf samples. Genecombination TAG (% dry weight) Ratio V2 + MGAT2 + DGAT1 1.54 ± 0.36 1.01V2 + MGAT2 + DGAT1 + GPAT4 1.56 ± 0.18

Example 15 Constitutive Expression of a MonoacylglycerolAcyltransferase, Diacycerol Acyltransferase, WRI1 Transcription Factorand AGPase-hpRNAi Silencing Construct in Plant Cells

A DNA fragment corresponding to nucleotides 595 to 1187 of the mRNAencoding the Nicotiana tabacum AGPase small subunit (DQ399915) (Kwak etal., 2007) was synthesized. The 593 bp 1118501_NtAGP fragment was firstcut with NcoI, treated with DNA polymerase I large (Klenow) fragment togenerate 5′ blunt ends and finally digested with XhoI. Similarly, thepENTR11-NCOI entry vector was first digested with BamHI, treated withDNA polymerase I large (Klenow) fragment and cut with XhoI. Ligation ofthe 1118501_NtAGP insert into pENTR11-NCOI generated thepENTR11-NCOI-NtAGP entry clone. LR recombination between thepENTR11-NCOI-NtAGP entry clone and the pHELLSGATE12 destination vectorgenerated pTV35, a binary vector containing the NtAGPase RNAi cassetteunder the control of the 35S promotor. Other constructs are described inExamples 11 and 12. Transient expression in N. benthamiana leaf tissuewas performed as described in Example 1.

Expression of the N. tabacum AGPase silencing construct together withthe genes coding for MGAT2 and WRI resulted in a 1.7-fold increase inleaf TAG levels as quantified by Iatroscan (Table 19). In the absence ofthe MGAT2 acyltransferase TAG levels dropped almost 3-fold. Thereforethe observed TAG increase cannot be attributed solely to the silencingof the endogenous N. benthamiana AGPase gene. Surprisingly, substitutingMGAT2 for the A. thaliana DGAT1 did not alter TAG levels in infiltratedN. benthamiana leaves in combination with the N. tabacum AGPasesilencing construct. Silencing of the N. benthamiana AGPase thereforeappears to have a different metabolic effect on MGAT and DGATacyltransferases. A similar difference is also observed in the maximumobserved TAG levels with WRI1 and the AGPase silencing construct incombination with MGAT2 or DGAT1 yielding 6.16% and 5.51% leaf oilrespectively (FIG. 12).

TABLE 19 TAG levels in triplicate Nicotiana benthamiana leaf tissuetransiently transformed with 35S:V2, 35S:MGAT2, 35S:DGAT1, 35S:WRI1 and35S:AGPase-hpRNAi TAG TAG Control (% dry Genes (% dry combinationweight) ¹ expressed weight) ¹ Ratio ² V2, WRI1, 2.33 ± 1.23 V2, WRI1,3.60 ± 0.98 1.69 ± 0.40 MGAT2 MGAT2, AGPase- hpRNAi V2, WRI1, 1.86 ±0.20 V2, WRI1, 5.21 ± 1.48 2.87 ± 1.01 AGPase- MGAT2, hpRNAi AGPase-hpRNAi V2, WRI1, 4.99 ± 0.95 V2, WRI1, 4.77 ± 0.79 0.96 ± 0.07 DGAT1DGAT1, AGPase- hpRNAi ¹ Average of three different infiltrated leaves asquantified by Iatroscan; ² Average ratio based on side-by-sidecomparisons on the same leaves

Overexpression of WRI1 and MGAT in combination with AGPase silencing isparticularly promising to increase oil yields in starch accumulatingtissues. Examples include tubers such as for potatoes, and the endospermof cereals, potentially leading to cereals with increased grain oilcontent (Barthole et al., 2011). Although N. tabacum and N. benthamianaAGPase genes are likely to bear significant sequence identity, it islikely that a N. benthamiana AGPase-hpRNAi construct will furtherelevate TAG yields due to improved silencing efficiency.

Additional data was obtained from a further experiment where leafsamples were pooled across leaves within same plant, 6 replicates ofeach. The data is shown in Tables 19 and 20.

TABLE 20 TAG content of infiltrated N. benthamiana leaf samples. GeneTAG (% dry combination weight) Ratio V2 + MGAT2 + DGAT1 + 1.93 ± 0.181.14 WRI1 + Oleosin V2 + MGAT2 + DGAT1 + 2.19 ± 0.19 WRI1 + Oleosin +AGPase-hpRNAi

Example 16 Constitutive Expression of a MonoacylglycerolAcyltransferase, Diacycerol Acyltransferase, WRI1 Transcription Factorand an Oleosin Protein in Plant Cells

A pRSh1 binary vector containing the gene coding for the S. indicum seedoleosin (Scott et al., 2010) under the control of the 35S promoter wasprovided by Dr. N. Roberts (AgResearch Limited, New Zealand). Otherconstructs are described in Examples 11 and 12. Transient expression inN. benthamiana leaf tissue was performed as described in Example 1.

When the sesame oleosin protein was expressed together with the A.thaliana WRI transcription factor and M. musculus MGAT2 acyltransferase,TAG levels in N. benthamiana leaves as quantified by Iatroscan werefound to be 2.2-fold higher (Table 21). No significant changes in theleaf TAG fatty acid profiles were detected (Table 22). A small increasein TAG was also observed when the A. thaliana DGAT1 acyltransferase wasincluded. Compared to the V2 negative control, the combined expressionof WRI1, DGAT1 and the sesame oleosin protein resulted in a 3-fold TAGincrease and a maximum observed TAG level of 7.72% (Table 21 and FIG.12). Leaf TAG levels were further elevated by a factor of 2.5 uponincluding the MGAT2 acyltransferase. This corresponded to an average of5.7% and a maximum observed of 18.8% TAG on a dry weight basis. Theadditional increase in leaf TAG when MGAT2 was included clearlydemonstrates the positive effect of this acyltransferase on thebiosynthesis and accumulation of neutral lipids in transgenic leaftissues.

The experiment was repeated with the combination of genes for expressingV2 and V2+MGAT2+DGAT1+WRI1+Oleosin, tested in different N. benthamianaplants, with samples pooled across leaves from the same plant and with12 replicate infiltrations for each. The data is shown in Table 23.Replicate samples were also pooled across leaves from same plant, with 6repeats for each infiltration: The data is shown in Tables 24 and 25.

Although infiltration of N. benthamiana leaves resulted in increasedlevels of leaf oil (TAG), no significant increase in the total lipidcontent was detected, suggesting that a redistribution of fatty acidsfrom different lipids pools into TAG was occurring. In contrast, whenthe MGAT2 gene was coexpressed with the DGAT1, WRI1 and oleosin genes,total lipids were increased 2.21-fold, demonstrating a net increase inthe synthesis of leaf lipids.

Example 17 Constitutive Expression of a MonoacylglycerolAcyltransferase, Diacylglycerol Acyltransferase, WRI1 TranscriptionFactor and a FAD2-hpRNAi Silencing Construct in Plant Cells

A N. benthamiana FAD2 RNAi cassette under the control of a 35S promoterwas obtained by LR recombination into the pHELLSGATE8 destination vectorto generate vector pFN033. Other constructs are described in Examples 11and 12.

TABLE 21 TAG levels in triplicate Nicotiana benthamiana leaf tissuetransiently transformed with 35S:V2, 35S:MGAT2, 35S:DGAT1, 35S:WRI1 and35S:Oleosin. TAG TAG Control (% dry Genes (% dry combination weight) ¹expressed weight) ¹ Ratio ² V2, WRI1, 1.77 ± 0.75 V2, WRI1, 3.34 ± 0.192.20 ± 1.10 MGAT2 MGAT2, Oleosin V2, WRI1, 1.31 ± 0.19 V2, WRI1, 2.36 ±1.10 1.79 ± 0.84 Oleosin MGAT2, Oleosin V2, WRI1, 4.82 ± 1.67 V2, WRI1,6.02 ± 1.57 1.32 ± 0.43 DGAT1 DGAT1, Oleosin V2, WRI1, 5.17 ± 1.87 V2,WRI1, 6.34 ± 1.74 1.25 ± 0.11 MGAT2, MGAT2, DGAT1 DGAT1, Oleosin V2,WRI1, 4.61 ± 1.83 V2, WRI1, 5.48 ± 1.39 1.24 ± 0.26 DGAT1, MGAT2,Oleosin DGAT1, Oleosin V2 1.46 ± 0.67 V2, WRI1, 3.71 ± 1.50 3.00 ± 1.63DGAT1, Oleosin V2 0.90 ± 0.43 V2, WRI1, 5.74 ± 0.22 7.45 ± 3.52 MGAT2,DGAT1, Oleosin ¹ Average of three different infiltrated leaves asquantified by Iatroscan ² Average ratio based on side-by-sidecomparisons on the same leaves

TABLE 22 TAG fatty acid profiles of triplicate Nicotiana benthamianaleaf tissue transiently transformed with 35S:V2, 35S:MGAT2, 35S:DGAT1,35S:WRI1 and 35S:Oleosin. V2, WRI1, V2, WRI1, V2, WRI1, MGAT2, Fatty V2,WRI1, DGAT1, MGAT2, DGAT1, acid DGAT1 Oleosin DGAT1 Oleosin C14:0 0.05 ±0.04 0.02 ± 0.04 0.05 ± 0.05 0.04 ± 0.04 C14:1^(Δ9) 0.14 ± 0.03 0.10 ±0.09 0 0 C16:0 30.64 ± 1.32  29.96 ± 1.23  25.53 ± 2.30  23.74 ± 1.83 C16:1^(Δ9) 0.39 ± 0.17 0.32 ± 0.33 0.19 ± 0.02 0.36 ± 0.10 C18:0 9.85 ±0.34 10.23 ± 0.20  8.50 ± 1.42 8.30 ± 0.73 C18:1^(Δ9) 38.17 ± 1.28 39.01 ± 1.87  35.14 ± 6.58  38.64 ± 5.12  C18:1^(Δ11) 0.66 ± 0.04 0.74 ±0.20 0.53 ± 0.06 0.56 ± 0.02 C18:2^(Δ9,12) 11.58 ± 0.52  11.53 ± 0.86 16.48 ± 1.40  15.75 ± 1.58  C18:3^(Δ9,12,15) 3.80 ± 0.32 3.97 ± 0.299.35 ± 0.74 9.61 ± 0.87 C20:0 2.50 ± 0.20 2.41 ± 0.15 2.17 ± 0.42 1.64 ±0.11 C22:0 1.33 ± 0.21 1.16 ± 0.11 1.22 ± 0.22 0.80 ± 0.04 C24:0 0.90 ±0.19 0.55 ± 0.48 0.85 ± 0.23 0.55 ± 0.06

TABLE 23 TAG content of infiltrated N. benthamiana leaf samples. GeneTAG (% dry combination weight) Ratio V2 0.19 ± 0.05 18.74 V2 + MGAT2 +DGAT1 + 3.56 ± 0.86 WRI1 + Oleosin

TABLE 24 TAG content of infiltrated N. benthamiana leaf samples. GeneTAG (% dry combination weight) Ratio V2 + MGAT2 + DGAT1 + 2.17 ± 0.300.79 WRI1 V2 + MGAT2 + DGAT1 + 2.11 ± 0.20 WRI1 + Oleosin V2 + MGAT20.32 ± 0.06 2.19 V2 + MGAT2 + Oleosin 0.70 ± 0.17

TABLE 25 Total fatty acid content of infiltrated N. benthamiana leafsamples. V2 3.12 ± 0.14 1 V2 + MGAT2 3.28 ± 0.33 1.05 V2 + MGAT2 +DGAT1 + 6.88 ± 0.37 2.21 WRI1 + Oleosin

The genes coding for the mouse monoacylglycerol acyltransferase MGAT2,A. thaliana diacylglycerol acyltransferase DGAT1, A. thaliana WRI1 and aN. benthamiana FAD2 Δ12-fatty acid desaturase hairpin RNAi construct(Wood et al., manuscript in preparation) were expressed in combinationin N. benthamiana leaf tissue using the transient expression system asdescribed in Example 1.

Similar changes were observed in the fatty acid compositions of TAG,polar lipids and TFA of N. benthamiana leaves infiltrated with WRI1,MGAT2, DGAT1 and the Fad2 silencing contruct (Tables 26-28). In allthree lipid fractions, oleic acid levels were further increased andreached almost 20% in polar lipids, 40% in TFA and more than 55% in TAG.This increase came mostly at the expense of linoleic acid reflecting thesilencing effect on the endoplasmic reticulum FAD2 Δ12-desaturase. LeafTAG also contained less α-linolenic acid while levels in TFA and polarlipids were unaffected.

When these experiments were repeated and the fatty acid compositionsdetermined for TAG, polar lipids and total lipids, the results (Table29) were consistent with the first experiment.

TABLE 26 TAG fatty acid profiles of triplicate Nicotiana benthamianaleaf tissue transiently transformed with 35S:V2, 35S:MGAT2, 35S:DGAT1,35S:WRI1 and 35S:FAD2-hpRNAi. V2, WRI1, MGAT2, V2, WRI1, DGAT1, FattyMGAT2, FAD2- acid V2 DGAT1 hpRNAi C14:1^(Δ9) 0.28 ± 0.48 0.14 ± 0.120.08 ± 0.13 C16:0 22.73 ± 0.40  22.63 ± 1.43  19.11 ± 1.62  C16:1^(Δ9) 00.28 ± 0.02 0.51 ± 0.11 C18:0 7.31 ± 1.44 5.27 ± 0.19 5.05 ± 0.11C18:1^(Δ9) 29.87 ± 11.91 32.21 ± 4.73  55.21 ± 1.31  C18:1^(Δ11) 0 0.80± 0.04 0.89 ± 0.04 C18:2^(Δ9,12) 13.36 ± 3.22  20.23 ± 3.36  3.61 ± 0.18C18:3^(Δ9,12,15) 25.03 ± 10.14 15.18 ± 0.89  12.03 ± 0.72  C20:0 0.99 ±0.86 1.38 ± 0.07 1.41 ± 0.04 C20:1^(Δ11) 0 0.39 ± 0.05 0.62 ± 0.02 C22:00 0.85 ± 0.04 0.83 ± 0.05 C24:0 0.44 ± 0.76 0.64 ± 0.08 0.66 ± 0.05

TABLE 27 Fatty acid profiles of polar lipids isolated from triplicateNicotiana benthamiana leaf tissue transiently transformed with 35S:V2,35S:MGAT2, 35S:DGAT1, 35S:WRI1 and 35S:FAD2-hpRNAi. V2, WRI1, MGAT2, V2,WRI1, DGAT1, Fatty MGAT2, FAD2- acid V2 DGAT1 hpRNAi C14:1^(Δ9) 0.13 ±0.23 0.17 ± 0.15 0 C16:0 15.00 ± 0.30  15.99 ± 0.14  15.28 ± 0.31 C16:1^(Δ9) 2.66 ± 0.28 1.97 ± 0.40 2.09 ± 0.16 C18:0 2.47 ± 0.14 2.05 ±0.18 1.95 ± 0.09 C18:1^(Δ9) 5.12 ± 2.22 10.57 ± 1.99  18.99 ± 0.76 C18:1^(Δ11) 0.28 ± 0.24 0.61 ± 0.01 0.67 ± 0.03 C18:2^(Δ9,12) 10.26 ±0.96  12.39 ± 1.33  5.20 ± 0.32 C18:3^(Δ9,12,15) 63.90 ± 1.18  55.70 ±1.26  55.65 ± 1.23  C20:0 0.09 ± 0.15 0.19 ± 0.16 0.08 ± 0.14C20:1^(Δ11) 0 0 0 C22:0 0 0.17 ± 0.15 0 C24:0 0.09 ± 0.15 0.19 ± 0.160.09 ± 0.16

TABLE 28 Fatty acid profiles of total lipids isolated from triplicateNicotiana benthamiana leaf tissue transiently transformed with 35S:V2,35S:MGAT2, 35S:DGAT1, 35S:WRI1 and 35S:FAD2-hpRNAi. V2, WRI1, MGAT2, V2,WRI1, DGAT1, Fatty MGAT2, FAD2- acid V2 DGAT1 hpRNAi C14:1^(Δ9) 0.53 ±0.08 0.27 ± 0.02 0.26 ± 0.02 C16:0 16.00 ± 1.05  19.70 ± 0.63  17.30 ±0.72  C16:1^(Δ9) 2.02 ± 0.62 0.24 ± 0.02 0.28 ± 0.02 C18:0 3.75 ± 0.254.33 ± 0.09 4.17 ± 0.03 C18:1^(Δ9) 11.12 ± 6.77  23.32 ± 4.09  40.37 ±2.24  C18:1^(Δ11) 0.46 ± 0.08 0.69 ± 0.03 0.75 ± 0.01 C18:2^(Δ9,12)11.14 ± 0.83  17.28 ± 2.34  4.56 ± 0.29 C18:3^(Δ9,12,15) 53.27 ± 7.34 32.43 ± 1.49  30.43 ± 1.34  C20:0 0.51 ± 0.16 0.93 ± 0.05 0.92 ± 0.03C20:1^(Δ11) 0 0.26 ± 0.03 0.40 ± 0.02 C22:0 0.83 ± 0.24 0.36 ± 0.06 0.37± 0.04 C24:0 0.38 ± 0.09 0.19 ± 0.03 0.20 ± 0.02

TABLE 29 Fatty acid composition of TAG, Polar lipids and total lipids ininfiltrated N. benthamiana leaf samples. TAG Polar lipids Total lipidsV2 + MGAT2 + V2 + MGAT2 + V2 + MGAT2 + V2 + MGAT2 + DGAT1 + V2 + MGAT2 +DGAT1 + WRI1 + V2 + MGAT2 + DGAT1 + WRI1 + DGAT1 + WRI1 + WRI1 +Oleosin + DGAT1 + Oleosin + FAD2- DGAT1 + Oleosin + FAD2- OleosinFAD2-hpRNAi WRI1 + Oleosin hpRNAi WRI1 + Oleosin hpRNAi C14:0 0.00 0.000.00 0.00 0.06 ± 0.03 0.02 ± 0.03 C14:1^(Δ9) 0.00 0.00 0.05 ± 0.12 0.000.24 ± 0.03 0.19 ± 0.10 C16:0 19.63 ± 0.53  16.95 ± 1.13  15.85 ± 1.17 16.42 ± 2.11  16.88 ± 0.92  15.45 ± 1.24  C16:1^(Δ13t) 0.00 0.15 ± 0.162.31 ± 0.38 1.61 ± 0.85 1.03 ± 0.23 0.85 ± 0.23 C16:3^(Δ7,12,15) 0.000.00 7.54 ± 0.40 7.42 ± 0.59 3.25 ± 0.47 2.79 ± 0.43 C18:0 6.64 ± 1.356.99 ± 0.43 2.88 ± 0.13 2.41 ± 1.21 5.36 ± 0.15 5.40 ± 0.18 C18:1^(Δ9)29.45 ± 3.65  53.97 ± 1.51  8.56 ± 2.04 19.68 ± 1.32  20.59 ± 3.52 39.27 ± 2.28  C18:1^(Δ11) 0.59 ± 0.29 0.68 ± 0.34 0.45 ± 0.22 0.40 ±0.31 0.59 ± 0.03 0.66 ± 0.05 C18:2^(Δ9,12) 23.47 ± 1.18  5.29 ± 0.3713.13 ± 0.32  4.72 ± 2.33 18.35 ± 0.70  5.56 ± 0.28 C18:3^(Δ9,12,15)18.32 ± 5.48  12.84 ± 0.60  49.04 ± 2.78  47.22 ± 5.40  30.03 ± 3.51 26.28 ± 3.07  C20:0 1.12 ± 0.56 1.56 ± 0.04 0.19 ± 0.21 0.11 ± 0.17 0.88± 0.06 1.03 ± 0.07 C20:1^(Δ11) 0.00 0.20 ± 0.22 0.00 0.00 0.08 ± 0.080.22 ± 0.11 C22:0 0.57 ± 0.28 0.81 ± 0.07 0.00 0.00 0.54 ± 0.05 0.61 ±0.07 C22:1 0.00 0.00 0.00 0.00 0.50 ± 0.03 0.22 ± 0.24 C22:2n6 0.00 0.000.00 0.00 0.80 ± 0.14 0.77 ± 0.13 C24:0 0.21 ± 0.32 0.57 ± 0.29 0.000.00 0.42 ± 0.01 0.48 ± 0.03 C24:1 0.00 0.00 0.00 0.00 0.42 ± 0.05 0.18± 0.2 

Example 18 Expression of Mus musculus MGAT1 and MGAT2 in Nicotianabenthamiana Cells by Stable Transformation

Constitutive Expression in N. benthamiana

The enzyme activity of the M. musculus MGAT1 and MGAT2 was demonstratedin Nicotiana benthamiana. The chimeric vectors 35S:Musmu-MGAT1 and35S:Musmu-MGAT2 were introduced into A. tumefaciens strain AGL1 viastandard electroporation procedure and grown on solid LB mediasupplemented with kanamycin (50 mg/L) and rifampicin (25 mg/L) andincubated at 28° C. for two days. A single colony was used to initiatefresh culture. Following 48 hours culturing with vigorous aeration, thecells were collected by centrifugation at 2,000×g and the supernatantwere removed. The cells were resuspended in a new solution containing50% LB and 50% MS medium at the density of OD₆₀₀=0.5. Leaf samples ofNicotiana benthamiana plants grown asceptically in vitro were excisedand cut into square sections around 0.5-1 cm² in size with a sharpscalpel while immersed in the A. tumefaciens solution. The wounded N.benthamiana leaf pieces submerged in A. tumefaciens were allowed tostand at room temperature for 10 min prior to being blotted dry on asterile filter paper and transferred onto MS plates without supplement.Following a co-cultivation period of two days at 24° C., the explantswere washed three times with sterile liquid MS medium, and finally blotdry with sterile filter paper and placed on the selectiveagar-solidified MS medium supplemented with 1.0 mg/L bencylaminopurine(BAP), 0.25 mg/L indoleacetic acid (IAA), 50 mg/L kanamycin and 250 mg/Lcefotaxime and incubated at 24° C. for two weeks to allow for shootdevelopment from the transformed N. benthamiana leaf discs. To establishin vitro transgenic plants, healthy green shoots were cut off andtransferred onto a new 200 mL tissue culture pots containingagar-solidified MS medium supplemented with 25 μg/L IAA and 50 mg/Lkanamycin and 250 mg/L cefotaxime.

Expression of the MGAT1 and MGAT2 transgenes was determined by Real-TimePCR. Highly-expressing lines were selected and their seed harvested.This seed was planted directly onto soil and the segregating populationof seedlings harvested after four weeks. Highly-expressing events wereselected and seed produced by these planted out directly onto soil toresult in a segregating population of 30 seedlings. After three weeksleaf discs were taken from each seedling for DNA extraction andsubsequent PCR to determine which lines were transgenic and which werenull for the transgene. The population was then harvested with theentire aerial tissue from each seedling cleaned of soil andfreeze-dried. The dry weight of each sample was recorded and totallipids isolated. The TAG in these total lipid samples was quantified byTLC-FID and the ratio of TAG to an internal standard (DAGE) in eachsample determined (FIG. 14). The average level of TAG in the transgenicseedlings of 35S:Musmu-MGAT2 line 3347-19 was found to be 4.1-foldhigher than the average level of TAG in the null seedlings. The eventwith the largest increase in TAG had 7.3-fold higher TAG than theaverage of the null events.

Constitutive Expression in A. thaliana

The enzyme activity of the M. musculus MGAT1 and MGAT2 was demonstratedin A. thaliana. The chimeric vectors 35S:Musmu-MGAT1 and 35S:Musmu-MGAT2along with the empty vector control pORE04 were transformed in A.thaliana by the floral dip method and seed from primary transformantsselected on kanamycin media. The T2 seed from these T1 plants washarvested and TFA of the seeds from each plant determined (FIG. 15). Theaverage mg TFA/g seed was found to be 139±13 for the control pORE04lines with median 136.0, 152±14 for the 35S:MGAT1 lines with median155.1 and 155±11 for the 35S:MGAT2 lines with median 154.7. Thisrepresented an average TFA increase compared to the control of 9.7% for35S:MGAT1 and 12.1% for 35S:MGAT2.

Example 19 Additional Genes

Further Increases in Oil

Additional genes are tested alongside the combinations described aboveto determine whether further oil increases can be achieved. Theseinclude the following Arabidopsis genes: AT4G02280, Sucrose synthaseSUSS; AT2G36190, Invertase CWINV4; AT3G13790, Invertase CWINV1;AT1G61800, Glucose 6 phosphate:phosphate translocator GPT2; AT5G33320,Phosphoenolpyruvate transporter PPT1; AT4G15530, Pyruvate orthophosphatedikinase Plastid-PPDK; AT5G52920, Pyruvate kinase pPK-β1. The genescoding for these enzymes are synthesised and cloned into theconstitutive binary expression vector pJP3343 as EcoRI fragments fortesting in N. benthamiana.

When a number of genes were added to the combination of WRI1, DGAT1,MGAT2 and oleosin and expressed in N. benthamiana leaves, no additionalincrease in the level of TAG was observed, namely for: safflower PDAT,Arabidopsis thaliana PDAT1, Arabidopsis thaliana DGAT2, Arabidopsisthaliana caleosin, peanut oleosin, Arabidopsis thaliana haemoglobin 2,Homo sapiens iPLAh, Arabidopsis thaliana GPAT4, E. coli G3Pdehydrogenase, yeast G3P dehydrogenase, castor LPAAT2, Arabidopsisthaliana beta-fructofuranosidase (ATBFRUCT1, NM_112232), Arabidopsisthaliana beta-fructofuranosidase (cwINV4, NM_129177), indicating thatnone of these enzyme activities were rate limiting in N. benthamianaleaves when expressed transiently. This does not indicate that they willhave no effect in stably-transformed plants, such as in seed, or inother organisms.

Further additional genes are tested for additive or synergistic oilincrease activity. These include the following Arabidopsis thaliana genemodels or their encoded proteins, and homologues from other species,which are grouped by putative function and have previously been shown tobe upregulated in tissues with increased oil content. Genes/proteinsinvolved in sucrose degradation: AT1G73370, AT3G43190, AT4G02280,AT5G20830, AT5G37180, AT5G49190, AT2G36190, AT3G13784, AT3G13790,AT3G52600. Genes/proteins involved in the oxidative pentose phosphatepathway: AT3G27300, AT5G40760, AT1G09420, AT1G24280, AT5G13110,AT5G35790, AT3G02360, AT5G41670, AT1G64190, AT2G45290, AT3G60750,AT1G12230, AT5G13420, AT1G13700, AT5G24410, AT5G24420, AT5G24400,AT1G63290, AT3G01850, AT5G61410, AT1G71100, AT2G01290, AT3 G04790,AT5G44520, AT4G26270, AT4G29220, AT4G32840, AT5G47810, AT5G56630,AT2G22480, AT5G61580, AT1G18270, AT2G36460, AT3G52930, AT4G26530,AT2G01140, AT2G21330, AT4G38970, AT3G55440, AT2G21170. Genes/proteinsinvolved in glycolysis: AT1G13440, AT3G04120, AT1G16300, AT1G79530,AT1G79550, AT3G45090, AT5G60760, AT1G56190, AT3G12780, AT5G61450,AT1G09780, AT3G08590, AT3G30841, AT4G09520, AT1G22170, AT1G78050,AT2G36530, AT1G74030. Genes/proteins which function as plastidtransporters: AT1G61800, AT5G16150, AT5G33320, AT5G46110, AT4G15530,AT2G36580, AT3G52990, AT3G55650, AT3G55810, AT4G26390, AT5G08570,AT5G56350, AT5G63680, AT1G32440, AT3G22960, AT3G49160, AT5G52920.Genes/proteins involved in malate and pyruvate metabolism: AT1G04410,AT5G43330, AT5G56720, AT1G53240, AT3G15020, AT2G22780, AT5G09660,AT3G47520, AT5G58330, AT2G19900, AT5G11670, AT5G25880, AT2G13560,AT4G00570.

Constructs are prepared which include sequences encoding these candidateproteins, which are infiltrated into N. benthamiana leaves as inprevious experiments, and the fatty acid content and compositionanalysed. Genes which aid in increasing non-polar lipid content arecombined with the other genes as described above, principally thoseencoding MGAT, Wri1, DGAT1 and an Oleosin, and used to transform plantcells.

Increases in Unusual Fatty Acids

Additional genes are tested alongside the combinations described aboveto determine whether increases in unusual fatty acids can be achieved.These include the following genes (provided are the GenBank AccessionNos.) which are grouped by putative function and homologues from otherspecies. Delta-12 acetylenases ABC00769, CAA76158, AAO38036, AAO38032;Delta-12 conjugases AAG42259, AAG42260, AAN87574; Delta-12 desaturasesP46313, ABS18716, AAS57577, AAL61825, AAF04093, AAF04094; Delta-12epoxygenases XP_001840127, CAA76156, AAR23815; Delta-12 hydroxylasesACF37070, AAC32755, ABQ01458, AAC49010; and Delta-12 P450 enzymes suchas AF406732.

Constructs are prepared which include sequences encoding these candidateproteins, which are infiltrated into N. benthamiana leaves as inprevious experiments, and the fatty acid content and compositionanalysed. The nucleotide sequences of the coding regions may becodon-optimised for the host species of interest. Genes which aid inincreasing unusual fatty acid content are combined with the other genesas described above, principally those encoding MGAT, WRI1, DGAT1 and anOleosin, and used to transform plant cells.

Example 20 Stable Transformation of Plants Including Nicotiana tabacumwith Combinations of Oil Increase Genes

An existing binary expression vector, pORE04+11ABGBEC (U.S. ProvisionalPatent Application No. 61/660,392), which contained a doubleenhancer-region 35S promoter expressing the NPTII kanamycin resistancegene and three gene expression cassettes, was used as a starting vectorto prepare several constructs each containing a combination of genes forstable transformation of plants. This vector was modified by exchangingthe expressed genes with oil increase genes, as follows. pORE04+11ABGBECwas first modified by inserting an intron-interrupted sesame oleosingene, flanked by Nod sites, from the vector pRSh1-PSP1 into thepORE04+11ABGBEC Nod sites to generate pJP3500. pJP33500 was thenmodified by inserting a codon-optimised DNA fragment encoding the A.thaliana WRL1 gene into the EcoRI sites to generate pJP3501. pJP3501 wasfurther modified by inserting a DNA fragment encoding the wild-type A.thaliana DGAT1 coding region, flanked by AsiSI sites, into the AsiSIsites to generate pJP3502 (SEQ ID NO:409). A final modification was madeby inserting another expression cassette, consisting of a doubleenhancer-region 35S promoter expressing a coding region encoding the M.musculus MGAT2, as a StuI-ZraI fragment into the SfoI site of pJP3502 togenerate pJP3503 (SEQ ID NO:410). The MGAT2 expression cassette wasexcised from pJP3347 at the StuI+ZraI sites. pJP3502 and pJP3503 wereboth used to stably transform N. tabacum as described below. By theseconstructions, pJP3502 contained the A. thaliana WRL1 and DGAT1 codingregions driven by the A. thaliana Rubisco small subunit promoter (SSU)and double enhancer-region 35S promoter, respectively, as well as aSSU:sesame oleosin cassette. The T-DNA region of this construct is shownschematically in FIG. 16. The vector pJP3503 additionally contained thee35S::MGAT2 cassette. This construct is shown schematically in FIG. 17.The nucleotide sequence of the T-DNA region of the construct pJP3503 isgiven as SEQ ID NO:412.

Stable Transformation of Nicotiana tabacum with Combinations of Genes

The binary vectors pJP3502 and pJP3503 were separately introduced intothe A. tumefaciens strain AGL1 by a standard electroporation procedure.Transformed cells were selected and grown on LB-agar supplemented withkanamycin (50 mg/l) and rifampicin (25 mg/l) and incubated at 28° C. fortwo days. A single colony of each was used to initiate fresh cultures inLB broth. Following 48 hours incubation with vigorous aeration, thecells were collected by centrifugation at 2,000 g and the supernatantwas removed. The cells were resuspended at the density of OD₆₀₀=0.5 infresh medium consisting of 50% LB and 50% MS medium.

Leaf samples of N. tabacum cultivar W38 grown asceptically in vitro wereexcised with a scalpel and cut into pieces of about 0.5-1 cm² in sizewhile immersed in the A. tumefaciens suspensions. The cut leaf pieceswere left in the A. tumefaciens suspensions at room temperature for 15minutes prior to being blotted dry on a sterile filter paper andtransferred onto MS plates without antibiotic supplement. Following aco-cultivation period of two days at 24° C., the explants were washedthree times with sterile, liquid MS medium, then blotted dry withsterile filter paper and placed on the selective MS agar supplementedwith 1.0 mg/L benzylaminopurine (BAP), 0.5 mg/L indoleacetic acid (IAA),100 mg/L kanamycin and 200 mg/L cefotaxime. The plates were incubated at24° C. for two weeks to allow for shoot development from the transformedN. tabacum leaf pieces.

To establish rooted transgenic plants in vitro, healthy green shootswere cut off and transferred to MS agar medium supplemented with 25 μg/LIAA, 100 mg/L kanamycin and 200 mg/L cefotaxime. After roots haddeveloped, individual plants were transferred to soil and grown in theglasshouse. Leaf samples were harvested at different stages of plantdevelopment including before and during flowering. Total fatty acids,polar lipids and TAG were quantified and their fatty acid profilesdetermined by TLC/GC as described in Example 1.

Analysis of pJP3503 Transformants

For the transformation with pJP3503 (“4-gene construct”), leaf samplesof about 1 cm² were taken from 30 primary transformants prior to flowerbuds forming and TAG levels in the samples were quantified by Iatroscan.Seven plants were selected for further analysis, of which fivedisplaying increased leaf oil levels and two exhibiting oil levelsessentially the same as wild-type plants. Freeze-dried leaf samples fromthese plants were analysed for total lipid content and TAG content andfatty acid composition by TLC and GC. Transformed plants numbered 4 and29 were found to have considerably increased levels of leaf oil comparedto the wild type, while plant number 21 exhibited the lowest TAG levelsat essentially wild-type levels (Table 30). Plants numbered 11, 15 and27 had intermediate levels of leaf oil. Oleic acid levels in TAG werefound to be inversely correlated to the TAG yields, consistent with theresults of the earlier transient expression experiments in N.benthamiana.

In the transformed plants numbered 4 and 29, leaf oil content (as apercentage of dry weight) was found to increase considerably at the timeof flowering (Table 31). From the data in Table 31, the increase was atleast 1.7- and 2.4-fold for plants 4 and 29, respectively. No suchchange was observed for plant 21 which had TAG levels similar to thewild-type control. Oleic acid levels in the TAG fractions isolated fromeach sample followed a similar pattern. This fatty acid accumulated upto 22.1% of the fatty acid in TAG from plants 4 and 29, a 17-18-foldincrease compared to plant 21 and the wild-type. The increase in oleicacid was accompanied by increased linoleic acid and palmitic acid levelswhile α-linolenic acid levels dropped 8-fold compared to in plant 21 andthe wild-type control. Unlike TAG, polar lipid levels decreased slightlyat the flowering stage in the three lines (Table 32). Changes in C18monounsaturated and polyunsaturated fatty acid levels in the polar lipidfractions of the three lines were similar to the shifts in their TAGcomposition although the changes in oleic acid and linoleic acid wereless marked. Significant increases in total leaf lipids were observedfor lines 4 and 29 during flowering with levels reaching more than 10%of dry weight (Table 33). Total leaf lipid levels in plant 21 before andduring flowering were similar to levels observed in wild-type plants atsimilar stages (Tables 33 and 35). Changes in the total lipid fatty acidcomposition of all three plants were similar to the respective TAG fattyacid compositions. Leaf oil in plant 4 during seed setting was found tobe further elevated at the onset of leaf chlorosis. The highest leaf TAGlevels detected at this stage corresponded to a 65-fold increasecompared to similar aged leaves in plant 21 during seed setting and a130-fold increase compared to similar leaves of flowering wild-typeplants (Table 34; FIG. 18).

The increased TAG in this plant coincided with elevated oleic acidlevels. Unlike plant 4, leaf TAG levels in the other two primarytransformants and wild-type tobacco did not increase, or only marginallyincreased, after flowering and during chlorosis. The lower leaves ofplants 4 and 29 exhibited reduced TAG levels upon senescence. In allplants, linoleic acid levels dropped while α-linolenic acid levels wereincreased with progressing leaf age.

Consistent with the increased TAG levels, total lipid levels in leavesof plants 4 and 29 during seed setting were further elevated compared tosimilar leaves of both plants during flowering (Tables 33 and 35). Thehighest total lipid level detected in plant 4 on a dry weight basis was15.8%, equivalent to a 7.6- and 11.2-fold increase compared to similarleaves of plant 21 and wild-type plants, respectively. Whilst the fattyacid composition of total lipid in the leaves of the wild type plant andplant 21 were similar, significant differences were observed in plants 4and 29. These changes mirrored those found in TAG of both primarytransformants.

Intriguingly, leaves of plants 4 and 29 before and during seed settingwere characterized by a glossy surface, providing a phenotypic changethat can serve as a phenotype that is easily scored visually, whichcould aid the timing of harvest for maximal oil content.

In summary, leaves of plants 4 and 29 rapidly accumulated TAG duringflowering up till seed setting. At the latter stage, the majority ofleaves exhibited TAG levels between 7% and 13% on a dry weight basis,compared to 0.1%-0.2% for line 21. These observed TAG levels and totallipid levels far exceed the levels achieved by Andrianov et al., (2010)who reported up to a maximum of 5.8% and 6.8% TAG in leaves of N.tabacum upon constitutive expression of the A. thaliana DGAT1 andinducible expression of the A. thaliana LEC2 genes.

TABLE 30 Percentage TAG (% weight of leaf dry weight) and oleic acidlevels (% of total fatty acids) in the TAG isolated from leaves ofselected primary tobacco plants transformed with pJP3503 Plant % TAGDevelopment No. (DW) % C18:1^(Δ9) stage of plant Wild type 0.06 2.3Budding Wild type 0.1 1.3 Flowering 3 0.05 1.5 Budding 4 1.29 10.2Budding 11 0.21 7.4 Flowering 15 0.23 4.5 No buds 21 0.01 1.9 No buds 270.19 3.3 Budding 29 1.15 10.4 No buds

TABLE 31 TAG levels (% weight of leaf dry weight) and fatty acidcomposition of TAG isolated from wild-type and three selected tobaccoplants transformed with pJP3503, before and during flowering. The datashown are averages and standard deviations of 2-3 independent repeats.Before flowering Flowering Wild type Plant 21 Plant 4 Plant 29 Wild typePlant 21 Plant 4 Plant 29 %/leaf DW 0.1 0.0 3.1 ± 0.3 4.1 ± 0.3 0.1 0.1± 0.0 7.3 ± 0.3 6.9 ± 0.5 C14:0 0.6 0.6 ± 0.2 0.1 ± 0.0 0.2 ± 0.0 0.50.4 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 C16:0 9.1 15.9 ± 0.3  42.0 ± 0.5  34.9 ±0.7  7.5 15.0 ± 0.7  33.1 ± 1.0  24.8 ± 1.4  C16:1^(Δ3t) 0.0 0.4 ± 0.10.1 ± 0.0 0.2 ± 0.0 0.4 0.2 ± 0.0 0.1 ± 0.0 0.2 ± 0.0 C16:1^(Δ9) 0.3 0.6± 0.0 3.3 ± 0.2 1.5 ± 0.1 0.3 0.6 ± 0.0 3.3 ± 0.2 1.2 ± 0.1C16:3^(Δ7,12,15) 3.7 0.8 ± 0.1 0.1 ± 0.0 0.2 ± 0.0 3.6 0.8 ± 0.1 0.1 ±0.0 0.2 ± 0.0 C18:0 3.2 4.6 ± 0.4 3.0 ± 0.1 5.6 ± 0.3 2.4 3.4 ± 0.0 3.3± 0.1 4.6 ± 0.1 C18:1^(Δ9) 2.3 1.2 ± 0.2 10.6 ± 0.3  10.6 ± 0.2  1.3 1.2± 0.2 19.1 ± 1.8  22.1 ± 2.7  C18:1^(Δ11) 0.1 0.1 ± 0.0 2.2 ± 0.2 1.2 ±0.1 0.1 0.1 ± 0.0 2.1 ± 0.0 0.9 ± 0.0 C18:2^(Δ9,12) 26.9 20.4 ± 1.1 20.7 ± 0.7  30.0 ± 1.0  23.7 19.5 ± 0.8  25.3 ± 0.6  34.7 ± 1.0 C18:3^(Δ9,12,15) 52.6 54.0 ± 0.8  15.9 ± 0.5  11.5 ± 1.3  59.3 57.5 ±0.4  10.8 ± 0.5  7.1 ± 0.6 C20:0 0.3 0.6 ± 0.0 1.0 ± 0.0 2.1 ± 0.2 0.30.4 ± 0.0 1.3 ± 0.0 2.0 ± 0.1 C20:1^(Δ11) 0.1 0.0 0.0 0.2 ± 0.1 0.1 0.1± 0.0 0.1 ± 0.0 0.3 ± 0.0 C20:2^(Δ11,14) 0.2 0.2 ± 0.0 0.0 0.1 ± 0.0 0.20.3 ± 0.0 0.0 0.1 ± 0.0 C20:3^(Δ11,14,17) 0.2 0.2 ± 0.0 0.0 0.0 0.1 0.2± 0.0 0.0 0.0 C22:0 0.1 0.2 ± 0.0 0.5 ± 0.0 1.0 ± 0.1 0.0 0.2 ± 0.0 0.7± 0.0 1.0 ± 0.0 C24:0 0.4 0.1 ± 0.1 0.4 ± 0.0 0.9 ± 0.1 0.3 0.1 ± 0.00.5 ± 0.0 0.7 ± 0.1

TABLE 32 Polar lipid levels (% weight of leaf dry weight) and fatty acidcomposition of polar lipids of leaf tissue from selected tobacco plantstransformed with pJP3503, before and during flowering. Data shown arethe average and standard deviations of 2-3 independent repeats, exceptfor plant 21 (before flowering). Before flowering Flowering Plant 21Plant 4 Plant 29 Plant 21 Plant 4 Plant 29 %/leaf DW 2.0 3.3 ± 0.4 2.5 ±0.4 1.7 ± 0.0 2.4 ± 0.0 1.7 ± 0.1 C14:0 0.0 0.0 0.0 0.0 0.0 0.0 C16:012.0 18.7 ± 0.6  12.7 ± 0.4  12.7 ± 0.0  16.2 ± 0.2  11.8 ± 0.2 C16:1^(Δ3t) 1.6 0.5 ± 0.1 1.4 ± 0.1 1.4 ± 0.0 1.1 ± 0.2 1.4 ± 0.1C16:1^(Δ9) 0.1 2.0 ± 0.1 0.7 ± 0.1 0.1 ± 0.0 1.8 ± 0.1 0.5 ± 0.0C16:3^(Δ7,12,15) 7.5 2.4 ± 0.2 3.8 ± 0.1 6.3 ± 0.0 1.0 ± 0.1 3.7 ± 0.3C18:0 2.1 1.1 ± 0.0 0.9 ± 0.1 2.5 ± 0.1 1.3 ± 0.0 1.4 ± 0.0 C18:1^(Δ9)1.1 5.6 ± 0.5 4.1 ± 0.0 1.0 ± 0.1 11.9 ± 1.1  9.2 ± 0.9 C18:1^(Δ11) 0.12.2 ± 0.1 1.0 ± 0.1 0.1 ± 0.0 2.1 ± 0.1 0.6 ± 0.0 C18:2^(Δ9,12) 12.519.8 ± 1.0  19.7 ± 1.4  13.6 ± 0.1  27.6 ± 0.6  28.8 ± 0.5 C18:3^(Δ9,12,15) 62.3 46.8 ± 0.6  55.1 ± 0.8  61.3 ± 0.1  36.0 ± 1.1 41.6 ± 0.6  C20:0 0.3 0.3 ± 0.0 0.2 ± 0.0 0.3 ± 0.0 0.4 ± 0.0 0.4 ± 0.0C20:1^(Δ11) 0.0 0.0 0.0 0.0 0.0 0.0 C20:2^(Δ11,14) 0.1 0.0 0.0 0.1 ± 0.00.0 0.0 C20:3^(Δ11,14,17) 0.1 0.0 0.0 0.1 ± 0.0 0.0 0.0 C22:0 0.2 0.2 ±0.0 0.2 ± 0.0 0.2 ± 0.0 0.3 ± 0.0 0.3 ± 0.0 C24:0 0.2 0.2 ± 0.0 0.3 ±0.0 0.2 ± 0.0 0.3 ± 0.0 0.3 ± 0.0

TABLE 33 Total lipid levels (% weight of leaf dry weight) and fatty acidcomposition of total lipids in leaves from tobacco plants transformedwith pJP3503, just before and during flowering. Data shown are theaverage of 2-3 leaf samples. Before flowering Flowering Plant 21 Plant 4Plant 29 Plant 21 Plant 4 Plant 29 %/leaf DW 2.4 ± 0.2 6.9 ± 0.5 4.9 ±1.1 2.0 ± 0.1 9.8 ± 0.3 8.8 ± 0.3 C14:0 0.1 ± 0.0 0.1 ± 0.0 0.1 ± 0.00.1 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 C16:0 12.1 ± 0.1  27.6 ± 1.2  20.7 ± 0.6 12.7 ± 0.1  26.9 ± 0.9  20.2 ± 1.2  C16:1^(Δ3t) 2.1 ± 0.2 0.4 ± 0.0 0.9± 0.1 2.7 ± 0.4 0.8 ± 0.1 0.7 ± 0.1 C16:1^(Δ9) 0.0 2.6 ± 0.2 1.0 ± 0.10.0 2.9 ± 0.1 1.0 ± 0.0 C16:3^(Δ7,12,15) 6.9 ± 0.3 1.3 ± 0.1 2.1 ± 0.15.6 ± 0.2 0.4 ± 0.0 0.9 ± 0.1 C18:0 2.2 ± 0.1 1.9 ± 0.0 2.8 ± 0.1 2.6 ±0.1 2.6 ± 0.1 3.6 ± 0.1 C18:1^(Δ9) 1.1 ± 0.2 7.5 ± 0.5 6.6 ± 0.0 1.1 ±0.2 16.2 ± 1.5  17.8 ± 2.0  C18:1^(Δ11) 0.2 ± 0.0 2.1 ± 0.1 1.1 ± 0.10.2 ± 0.0 2.0 ± 0.0 0.8 ± 0.0 C18:2^(Δ9,12) 13.8 ± 1.5  21.4 ± 0.7  26.5± 1.6  14.1 ± 0.5  27.3 ± 0.4  35.9 ± 0.5  C18:3^(Δ9,12,15) 60.6 ± 1.4 33.6 ± 1.5  36.0 ± 1.0  58.5 ± 1.2  18.3 ± 0.5  15.5 ± 0.6  C20:0 0.3 ±0.0 0.7 ± 0.0 1.0 ± 0.1 0.3 ± 0.0 1.0 ± 0.0 1.5 ± 0.0 C20:1^(Δ11) 0.00.0 0.1 ± 0.0 0.0 0.1 ± 0.0 0.3 ± 0.0 C20:2^(Δ11,14) 0.1 ± 0.0 0.0 0.1 ±0.0 0.2 ± 0.0 0.0 0.0 C20:3^(Δ11,14,17) 0.1 ± 0.0 0.0 0.0 0.3 ± 0.0 0.6± 0.0 0.8 ± 0.0 C22:0 0.2 ± 0.0 0.3 ± 0.0 0.5 ± 0.1 1.3 ± 0.1 0.3 ± 0.00.2 ± 0.0 C24:0 0.2 ± 0.0 0.3 ± 0.0 0.5 ± 0.0 0.3 ± 0.0 0.5 ± 0.0 0.6 ±0.0

TABLE 34 TAG levels (% weight of leaf dry weight) and fatty acidcomposition of TAG isolated from different aged leaves, post flowering,of three selected tobacco plants transformed with pJP3503. Plant:^(a)Wild type Plant 21 Plant 4 Plant 29 Leaf stage^(b) G YG G YG G YG Y G YGY^(c) %/leaf DW 0.1 0.1 0.1 0.2 9.5 13.0 10.7 7.0 7.1 2.1 C14:0 0.5 0.30.4 0.3 0.2 0.1 0.1 0.2 0.2 0.3 C16:0 7.5 14.8 8.9 14.9 31.1 33.3 38.025.7 33.0 38.5 C16:1^(Δ3t) 0.4 0.3 0.3 0.2 0.1 0.1 0.1 0.2 0.2 0.3C16:1^(Δ9) 0.3 0.2 0.2 0.2 3.0 3.1 2.4 1.2 1.1 0.7 C16:3^(Δ7,12,15) 3.60.6 3.2 1.2 0.1 0.1 0.1 0.3 0.3 0.2 C18:0 2.4 3.3 2.7 3.9 3.2 2.8 2.84.6 4.5 4.1 C18:1^(Δ9) 1.3 0.8 2.3 0.7 17.6 21.2 21.7 16.4 11.1 10.7C18:1^(Δ11) 0.1 0.1 0.1 0.1 2.1 1.8 1.5 0.9 0.8 0.7 C18:2^(Δ9,12) 23.717.5 24.3 15.2 28.2 22.0 17.0 36.5 32.6 24.6 C18:3^(Δ9,12,15) 59.3 60.956.4 62.2 11.3 12.8 13.2 9.5 11.9 14.8 C20:0 0.3 0.4 0.3 0.5 1.4 1.2 1.32.1 2.1 2.1 C20:1^(Δ11) 0.1 0.1 0.1 0.0 0.1 0.1 0.1 0.3 0.2 0.1C20:2^(Δ11,14) 0.2 0.2 0.2 0.1 0.1 0.0 0.0 0.1 0.1 0.0 C20:3^(Δ11,14,17)0.1 0.2 0.1 0.1 0.0 0.0 0.0 0.0 0.0 0.0 C22:0 0.0 0.1 0.1 0.1 0.8 0.70.9 1.2 1.2 1.5 C24:0 0.3 0.2 0.2 0.2 0.7 0.5 0.7 0.9 0.8 1.4 ^(a)Leafsamples were taken from wild type at flowering stage and from the threepJP3503 primary transformants during seed setting. ^(b)leaf stages bycolour indicated by ‘G’, green; ‘YG’, yellow-green; ‘Y’, yellow ^(c)veryold leaf

TABLE 35 Total lipid yield (% weight of leaf dry weight) and fatty acidcomposition of total lipid isolated from differently aged leaves ofwild-type and three selected tobacco plants transformed with pJP3503.Plant: Wild type Plant 21 Plant 4 Plant 29 Leaf stage^(a) G^(b) G^(c) YGG YG G YG Y G YG Y %/leaf DW 2.4 1.8 1.4 2.3 2.1 11.6 15.8 13.0 10.1 8.83.7 C14:0 0.1 0.2 0.1 0.2 0.1 0.2 0.1 0.1 0.1 0.1 0.2 C16:0 11.6 11.916.0 11.9 13.7 26.6 30.0 34.6 21.0 28.0 29.4 C16:1^(Δ3t) 4.1 6.3 3.2 3.12.0 0.5 0.4 0.5 0.8 0.6 0.9 C16:1^(Δ9) 0.0 0.0 0.0 0.0 0.0 3.0 2.9 2.31.2 1.1 0.7 C16:3^(Δ7,12,15) 6.7 5.3 3.6 5.6 5.3 0.4 0.3 0.3 1.0 0.9 1.5C18:0 2.4 2.9 4.1 3.0 3.2 2.9 2.6 2.8 3.7 4.0 3.7 C18:1^(Δ9) 1.4 1.2 0.91.4 0.5 15.8 20.2 20.8 13.6 10.2 11.3 C18:1^(Δ11) 0.5 0.9 0.4 0.4 0.22.1 1.9 1.5 0.9 0.7 0.7 C18:2^(Δ9,12) 16.0 15.5 16.5 15.6 12.4 28.9 23.318.6 34.6 33.4 26.8 C18:3^(Δ9,12,15) 54.4. 51.4 52.3 57.0 60.9 16.7 15.815.3 19.1 17.1 20.3 C20:0 0.5 0.6 0.8 0.4 0.5 1.2 1.0 1.3 1.6 1.7 1.6C20:1^(Δ11) 0.1 0.0 0.0 0.0 0.0 0.1 0.1 0.1 0.2 0.2 0.1 C20:2^(Δ11,14)0.1 0.1 0.1 0.1 0.1 0.0 0.0 0.0 0.1 0.1 0.0 C20:3^(Δ11,14,17) 0.1 0.10.1 0.1 0.1 0.0 0.0 0.0 0.0 0.0 0.0 C22:0 0.3 0.4 0.5 0.2 0.3 0.7 0.70.9 0.9 1.1 1.3 C24:0 0.3 0.3 0.4 0.2 0.3 0.6 0.5 0.7 0.7 0.8 1.2^(a)samples taken from plants harbouring multiple seed pods unlessindicated otherwise, ^(b)before flowering, ^(c)during floweringAnalysis of Tobacco Plants Transformed with pJP3502

For the transformation with pJP3502 (“3 gene construct”), the sucroselevel in the MS agar medium was reduced to half the standard level untilsufficient calli were established, which aided the recovery oftransformants expressing WRI1. Forty-one primary transformants wereobtained from the transformation with pJP3502 and transferred to thegreenhouse. Leaf samples of different age were collected at eitherflowering or seed setting stages (Table 36). The plants lookphenotypically normal except for three transformants, originating fromthe same callus in the transformation procedure and therefore likely tobe from the same transformation event, which were slightly smaller anddisplayed a glossy leaf phenotype similar to that observed for plant 4with pJP3503 (above) but less in extent.

Leaf disk samples of primary transformants were harvested duringflowering and TAG was quantified visualized by iodine staining afterTLC. Selected transgenic plants displaying increased TAG levels comparedto the wild type controls were further analyzed in more detail by TLCand GC. The highest TAG level in young green leaves was detected in line8.1 and corresponded to 8.3% TAG on a dry weight basis or an approximate83-fold increase compared to wild type leaves of the same age (Table36). Yellow-green leaves typically contained a higher oil contentcompared to younger green leaves with maximum TAG levels observed inline 14.1 (17.3% TAG on a dry weight basis). Total lipid content andfatty acid composition of total lipid in the leaves was also quantitated(Table 37).

Seed (T1 seed) was collected from the primary transformants at seedmaturity and some were sown to produce T1 plants. These plants werepredicted to be segregating for the transgene and therefore some nullsegregants were expected in the T1 populations, which could serve asappropriate negative controls in addition to known wild-type plantswhich were grown at the same time and under the same conditions. 51 T1plants, derived from primary transformant 14.1 which had a single-copyT-DNA insertion, which were 6-8 weeks of age and 10-25 cm in height wereanalysed together with 12 wild-type plants. The plants appearedphenotypically normal, green and healthy, and did not appear smallerthan the corresponding wild-type plants. Leaf samples of about 1 cmdiameter were taken from fully expanded green leaves. 30 of the T1plants showed elevated TAG levels in the leaves, of which 8 plantsshowed high levels of TAG, about double the level of TAG compared to theprimary transformant 14.1 at the same stage of plant development. Theselatter plants are likely to be homozygous for the transgenes. The levelof TAG and the TAG fatty acid composition in leaves of selected T1plants were measured by loading lipid isolated from about 5 mg dryweight of leaf tissue onto each TLC lane, the data is shown in Table 38.

TABLE 36 TAG levels (% weight of leaf dry weight) and fatty acidcomposition of TAG isolated from green leaves from ten selected tobaccoplants transformed with pJP3502. Line 2.1a 8.1 8.1 10.1 10.1 10.2 10.213.4 14.1 14.1 14.2 14.2 14.3 14.3 19.1 19.1 19.2 19.2 Stageb Y F Y F YS Y F S Y S Y F Y F Y F Y TAG 2.5 8.3 7.2 6.2 11.8 6.4 7.6 5.7 4.6 17.32.5 12.6 3 2 5.1 5.5 4.3 3.2 (% dry weight) C14:0 0.1 0.1 0.1 0.1 0.10.1 0.1 0.2 0.2 0.1 0.2 0.1 0.1 0.2 0.1 0.1 0.2 0.1 C16:0 26.4 28.5 31.422.6 24.0 26.9 29.7 28 28.2 28.6 20.1 27.3 19.4 30.4 23.2 34.5 25.4 36.7C16:1 Δ3 0.4 0.3 0.4 0.3 0.3 0.3 0.4 0.3 0.3 0.4 0.5 0.3 0.2 0.0 0.3 0.40.3 0.2 C16:1 Δ9 0.6 1.8 1.0 1.4 1.1 1.4 1.0 2 1.7 1.6 1.1 1.3 2.2 2.91.4 0.9 1.3 0.8 C16:3 Δ7,12,15 0.3 0.4 0.4 0.3 0.4 0.3 0.4 0.2 0.3 0.20.5 0.3 0.3 0.1 0.2 0.3 0.4 0.3 C18:0 7.1 3.8 4.0 3.8 3.5 4 4.0 4.2 4.14.1 4.5 3.9 3.3 3.0 4 4.2 4.3 4.3 C18:1 Δ9 9.6 18.1 11.2 33.1 25.1 21.310.4 24.8 18.6 25.8 16.1 17.3 14 1.9 25.5 8.5 20.2 10.4 C18:1 Δ11 0.51.2 0.8 1.3 0.8 1 0.7 1.2 1.1 1.0 0.8 0.8 1 0.7 1.3 0.6 1.4 0.7 C18:2Δ9,12 31.3 30.5 32.0 28 31.3 32.3 37.3 25.8 30.7 24.3 37.3 33.8 35.515.3 33.5 30.1 33.5 20.8 C18:3 Δ9,12,15 17.9 10.8 13.2 5.9 10.2 8.1 10.28.8 9.4 9.8 14.6 10.2 21.5 44.0 6.5 15.7 8.5 21.1 C20:0 3 2 2.3 1.5 1.62 2.5 2 2.4 2.0 2.1 2.2 1.2 0.9 1.7 2.2 2 2.0 C20:1 Δ11 0.3 0.3 0.3 0.30.0 0.3 0.3 0.3 0.4 0.0 0.5 0.0 0.3 0.0 0.4 0.0 0.4 0.2 C20:2 Δ11,14 0.10.1 0.1 0 0.1 0.1 0.1 0 0.1 0.1 0 0.1 0.1 0.0 0.1 0.0 0.1 0.0 C20:3Δ11,14,17 0 0 0.0 0 0.0 0 0.0 0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 C22:0 1.61.2 1.7 0.8 1.0 1.2 1.8 1.1 1.5 1.1 1 1.4 0.5 0.3 0.9 1.4 1.1 1.2 C24:00.9 0.9 1.1 0.6 0.7 0.8 1.2 0.9 1.1 1.0 0.8 1.1 0.4 0.3 0.7 1.1 0.9 1.1^(a)very old plant containing only yellow leaves ^(b)seed setting (‘S’),flowering (‘F’), yellow or yellowing leaves (‘Y’)

TABLE 37 Total lipid and fatty acid composition of total lipid extractedfrom yellow leaves from selected tobacco plants transformed with pJP3502(DGAT1 + WRI1 + Oleosin) Line C14:0 C16:0 C16d3 16:1w13t C16:1 C16:3C18:0 C18:1 C18:1d11 C18:2 14.1 0.1 26.6 0.0 0.6 1.6 0.3 3.8 25.6 1.125.2 14.2 0.1 25.2 0.0 0.6 1.3 0.4 3.6 16.5 0.8 35.1 10.1 0.1 22.4 0.00.7 1.3 0.6 3.2 24.3 0.8 31.9 13.4 0.2 19.6 0.0 3.6 0.0 1.7 4.1 3.9 0.634.6 Line C18:3n3 DW C20:0 C20:1d11 C20:2n6 C20:3n3 C22:0 C24:0 mg/100mg 14.1 11.0 1.8 0.3 0.1 0.0 1.0 0.9 23.4 14.2 11.6 1.9 0.3 0.1 0.0 1.51.0 15.5 10.1 11.4 1.5 0.3 0.0 0.0 0.9 0.6 9.7 13.4 27.7 1.6 0.3 0.0 0.01.5 0.8 2.0

TABLE 38 TAG levels (% weight of leaf dry weight) and fatty acidcomposition of TAG isolated from green leaves from selected tobacco T1plants transformed with pJP3502. Line No. 16:0 16:1 18:0 18:1d9 18:1d1118:2 18:3n3 20:0 22:0 24:0 % TAG 4.1 23.3 0.9 3.3 11.3 1.5 44.8 10.7 1.91.4 0.9 3.7 4.2 22.4 0.9 3.1 13.3 1.5 44.3 10.5 1.8 1.3 0.9 6.1 4.3 24.21.0 3.4 14.9 1.6 41.8 9.0 1.9 1.3 0.9 4.0 6.1 24.6 0.9 3.9 16.5 1.4 33.414.4 2.1 1.5 1.3 3.5 6.2 22.8 0.8 3.8 18.6 1.4 32.7 15.1 2.1 1.5 1.1 3.46.3 26.6 1.0 4.2 16.2 1.5 31.0 15.6 2.3 1.6 0.0 2.3 8.1 27.0 1.0 4.818.3 1.5 27.1 15.9 2.6 1.9 0.0 1.5 8.2 24.2 1.0 4.3 19.0 1.4 27.6 16.72.5 1.8 1.5 2.2 8.3 26.5 1.3 4.8 22.0 1.7 24.9 16.2 2.6 0.0 0.0 1.2 13.133.7 1.3 5.0 7.4 1.3 34.2 14.2 2.8 0.0 0.0 1.2 13.2 29.1 1.1 4.4 7.2 1.337.2 17.1 2.6 0.0 0.0 1.6 13.3 34.3 0.0 5.5 6.9 0.0 36.4 13.7 3.2 0.00.0 0.8 21.1 27.4 0.7 4.2 8.5 1.2 37.1 15.4 2.4 1.6 1.4 2.1 21.2 29.70.9 4.5 9.1 1.3 36.3 15.9 2.3 0.0 0.0 1.6 21.3 27.1 0.8 4.3 12.7 1.437.1 13.0 2.2 1.4 0.0 2.4 29.1 27.2 0.8 4.3 12.8 1.1 34.9 14.4 2.1 1.41.0 3.9 29.2 26.9 1.0 4.1 14.7 1.2 35.3 13.0 2.0 1.2 0.8 3.8 29.3 25.71.4 4.1 18.4 1.2 35.7 11.0 1.6 1.0 0.0 3.7 23.1 29.9 0.9 4.3 8.1 1.235.0 18.4 2.1 0.0 0.0 1.6 23.2 30.8 1.0 4.8 9.3 1.3 33.5 17.2 2.3 0.00.0 1.5 23.3 29.2 0.9 4.3 9.1 1.3 36.2 15.2 2.3 1.5 0.0 2.3 49.1 27.00.9 3.9 5.8 1.4 43.8 11.0 2.5 2.1 1.5 2.4 49.2 27.5 0.9 3.8 7.1 1.5 44.710.2 2.4 2.0 0.0 2.2

Genetic constructs suitable for transformation of monocotyledonousplants are made by exchanging the Arath-SSU promoters in pJP3502 andpJP3503 for promoters more active in monocots. Suitable promotersinclude constitutive viral promoters from monocot viruses or promotersthat have demonstrated to function in a transgenic context in monocotspecies (e.g., the maize Ubi promoter described by Christensen et al.,1996). Similarly, the CaMV-35S promoters in pJP3502 and pJP3503 areexchanged for promoters that are more active in monocot species. Theseconstructs are transformed into wheat, barley and maize using standardmethods.

Miscanthus Species

Genetic constructs for Miscanthus species transformation are made byexchanging the Arath-SSU promoters in pJP3502 and pJP3503 for promotersmore active in Miscanthus. Suitable promoters include constitutive viralpromoters, a ubiquitin promoter (Christensen et al., 1996) or promotersthat have demonstrated to function in a transgenic context inMiscanthus. Similarly, the CaMV-35S promoters in pJP3502 and pJP3503 areexchanged for promoters that are more active in Miscanthus. Newconstructs are transformed in Miscanthus by a microprojectile-mediatedmethod similar to that described by Wang et al., 2011.

Switchgrass (Panicum virgatum)

Genetic constructs for switchgrass transformation are made by exchangingthe Arath-SSU promoters in pJP3502 and pJP3503 for promoters more activein switchgrass. Suitable promoters include constitutive viral promotersor promoters that have demonstrated to function in a transgenic contextin switchgrass (e.g., Mann et al., 2011). Similarly, the CaMV-35Spromoters in pJP3502 and pJP3503 are exchanged for promoters that aremore active in switchgrass. New constructs are transformed inswitchgrass by an Agrobacterium-mediated method similar to thatdescribed by Chen et al., 2010 and Ramamoorthy and Kumar, 2012.

Sugarcane

Genetic constructs for sugarcane transformation are made by exchangingthe Arath-SSU promoters in pJP3502 and pJP3503 for promoters more activein sugarcane. Suitable promoters include constitutive viral promoters orpromoters that have demonstrated to function in a transgenic context insugarcane (e.g., the maize Ubi promoter described by Christensen et al.,1996). Similarly, the CaMV-35S promoters in pJP3502 and pJP3503 areexchanged for promoters that are more active in sugarcane. Newconstructs are transformed in sugarcane by a microprojectile-mediatedmethod similar to that described by Bower et al., 1996.

Elephant Grass

Genetic constructs for Pennisetum purpureum transformation are made byexchanging the Arath-SSU promoters in pJP3502 and pJP3503 for promotersmore active in elephant grass. Suitable promoters include constitutiveviral promoters or promoters that have demonstrated to function in atransgenic context in Pennisetum species such as P. glaucum like (e.g.the maize Ubi promoter described by Christensen et al., 1996).Similarly, the CaMV-35S promoters in pJP3502 and pJP3503 are exchangedfor promoters that are more active in Pennisetum species. New constructsare transformed in P. purpureum by a microprojectile-mediated methodsimilar to that described by Girgi et al., 2002.

Lolium

Genetic constructs for Lolium perenne and other Lolium speciestransformation are made by exchanging the Arath-SSU promoters in pJP3502and pJP3503 for promoters more active in ryegrass. Suitable promotersinclude constitutive viral promoters or promoters that have demonstratedto function in a transgenic context in Lolium species (e.g. the maizeUbi promoter described by Christensen et al., 1996). Similarly, theCaMV-35S promoters in pJP3502 and pJP3503 are exchanged for promotersthat are more active in Pennisetum species. New constructs aretransformed in Lolium perenne by a silicon carbide-mediated methodsimilar to that described by Dalton et al., 2002 or anAgrobacterium-mediated method similar to that described by Bettany etal., 2003.

pJP3502 and pJP3503 are modified to seed-specific expression geneticconstructs by exchanging the CaMV-35S and Arath-SSU promoters (exceptthe selectable marker cassette) with seed-specific promoters active inthe target species.

Canola

Genetic constructs for Brassica napus transformation are made byexchanging the CaMV-35S and Arath-SSU promoters in pJP3502 and pJP3503for promoters more active in canola. Suitable promoters includepromoters that have previously been demonstrated to function in atransgenic context in Brassica napus (e.g., the A. thaliana FAE1promoter, Brassica napus napin promoter, Linum usitatissimum conlinin1and conlinin2 promoters). New constructs are transformed in B. napus aspreviously described.

Soybean (Glycine max)

A genetic construct is made by cloning the PspOMI fragment from asynthesised DNA fragment having the nucleotide sequence shown in SEQ IDNO:415 (Soybean synergy insert; FIG. 19A) into a binary vector such aspORE04 at the Nod site. This fragment contains Arath-WRI1 expressed by aArath-FAE1 promoter, Arath-DGAT1 expressed by a Linus-Cnl2 promoter,Musmu-MGAT2 expressed by Linus-Cnl1 and Arath-GPAT4 expressed byLinus-Cnl1. A further genetic construct is made by exchanging the GPATcoding region for an oleosin coding region. A further genetic constructis made by deleting the MGAT expression cassette.

A genetic construct, pJP3569 (FIG. 21), was generated by cloning theSM-PstI fragment from the DNA molecule having the nucleotide sequenceshown in SEQ ID NO:415 into the PstI site of pORE04. This constructcontained (i) a coding region encoding the A. thaliana WRI1transcription factor, codon optimised for G. max expression, andexpressed from the G. max kunitz trypsin inhibitor 3 (Glyma-KTi3)promoter, (ii) a coding region encoding the Umbelopsis ramanniana DGAT2A(codon optimised as described by Lardizabal et al., 2008) and expressedfrom the G. max alpha-subunit beta-conglycinin (Glyma-b-conglycinin)promoter and (iii) a coding region encoding the M. musculus MGAT2, codonoptimised for G. max expression. A second genetic construct, pJP3570,was generated by cloning the SbfI-SwaI fragment of the DNA moleculehaving the nucleotide sequence shown in SEQ ID NO:415 into pORE04 at theEcoRV-PstI sites to yield a binary vector containing genes expressingthe A. thaliana WRI1 transcription factor and U. ramanniana DGAT2Aenzyme. Similarly, a third genetic construct, pJP3571, was generated bycloning the AsiSI fragment of the DNA molecule having the nucleotidesequence shown in SEQ ID NO:415 into the AsiSI site of pORE04 to yield abinary vector containing a gene encoding the U. ramanniana DGAT2Aenzyme. A fourth genetic construct, pJP3572, was generated by cloningthe NotI fragment of the DNA molecule having the nucleotide sequenceshown in SEQ ID NO:415 into pORE04 at the NotI site to yield a binaryvector containing a gene expressing the A. thaliana WRI1 transcriptionfactor. A fifth genetic construct, pJP3573, was generated by cloning theSwaI fragment of the DNA molecule having the nucleotide sequence shownin SEQ ID NO:415 into pORE04 at the EcoRV site to yield a binary vectorcontaining the gene encoding M. musculus MGAT2.

A sixth genetic construct, pJP3580, is generated by replacing the M.musculus MGAT2 with the Sesamum indicum oleosin gene.

Each of these six constructs are used to transform soybean, using themethods as described in Example 6. Transgenic plants produced by thetransformation with each of the constructs, particularly pJP3569,produce seeds with increased oil content.

Sugarbeet

The vectors pJP3502 and pJP3503 (see above) as used for thetransformation of tobacco are used to transform plants of sugarbeet(Beta vulgaris) by Agrobacterium-mediated transformation as described byLindsey & Gallois (1990). The plants produce greatly increased levels ofTAG in their leaves, similar in extent to the tobacco plants produced asdescribed above. Transgenic sugarbeet plants are harvested while theleaves are still green or preferably green/yellow just prior tobeginning of senescence or early in that developmental process, i.e.,and while the sugar content of the beets is at a high level and afterallowing accumulation of TAG in the leaves. This allows the productionof dual-purpose sugarbeets which are suitable for production of bothsugar from the beets and lipid from the leaves; the lipid may beconverted directly to biodiesel fuel by crushing the leaves andcentrifugation of the resultant material to separate the oil fraction,or the direct production of hydrocarbons by pyrolosis of the leafmaterial.

Promoters that are active in the root (tuber) of sugarbeet are also usedto express transgenes in the tuber.

Example 21 Stable Transformation of Solanum tuberosum with Oil IncreaseGenes

pJP3502, the intermediate binary expression vector described in theprevious example, was modified by first excising one SSU promoter byAscI+NcoI digestion and replacing it with the potato B33 promoterflanked by AscI and NcoI to generate pJP3504. The SSU promoter inpJP3504 along with a fragment of the A. thaliana WRL1 gene was replacedat the PspOMI sites by a potato B33 promoter with the same A. thalianaWRL1 gene fragment flanked by NotI-PspOMI to generate pJP3506. ThepJP3347 was added to pJP3506 as described in the above example togenerate pJP3507. This construct is shown schematically in FIG. 20. Itssequence is given in SEQ ID NO:413. The construct is used to transformpotato (Solanum tuberosum) to increase oil content in tubers.

Example 22 GPAT-MGAT Fusion Enzymes

The enzyme activity of GPAT-MGAT enzyme fusions is tested to determinewhether this would increase the accessibility of the GPAT-produced MAGfor MGAT activity. A suitable linker region was first synthesised andcloned into a cloning vector. This linker contained suitable sites forcloning the N-terminal (EcoRI-ZraI) and C-terminal coding regions(NdeI-SmaI or NdeI-PstI).atttaaatgcggccgcgaattcgtcgattgaggacgtccctactagacctgctggacctcctcctgctacttactacgattctctcgctgtgcatatggtcagtcatgcccgggcctgcaggcggccgcatttaaat (SEQ ID NO:414)

A GPAT4-MGAT2 fusion (GPAT4 N-terminus and MGAT2 C-terminus) was made byfirst cloning a DNA fragment encoding the A. thaliana GPAT4, flanked byMfeI and ZraI sites and without a C-terminal stop codon, into theEcoRI-ZraI sites. The DNA fragment encoding the M. musculus MGAT2,flanked by NdeI-PstI sites, was then cloned into the NdeI-PstI sites togenerate a single GPAT4-MGAT2 coding sequence. The fused coding sequencewas then cloned as a NotI fragment into pYES2 to generatepYES2::GPAT4-MGAT2 and the constitutive binary expression vector pJP3343to generate pJP3343::GPAT4-MGAT2.

Similarly, a MGAT2-GPAT4 fusion (MGAT2 N-terminus and GPAT4 C-terminus)was made by first cloning the DNA fragment encoding M. musculus MGAT2,flanked by EcoRI and ZraI sites without a C-terminal stop codon, intothe EcoRI-ZraI sites. The DNA fragment encoding the A. thaliana GPAT4,flanked by NdeI-PstI sites, was then cloned into the NdeI-PstI sites togenerate a single MGAT2-GPAT4 coding sequence. The fused coding sequencewas then cloned as a Nod fragment into pYES2 to generatepYES2::MGAT2-GPAT4 and the constitutive binary expression vector pJP3343to generate pJP3343::MGAT2-GPAT4.

The yeast expression vectors are tested in yeast S. cerevisiae and thebinary vectors are tested in N. benthamiana and compared for oil contentand composition with single-coding region controls.

Example 23 Discovery of Novel WRL1 Sequences

Three novel WRL1 sequences are cloned into pJP3343 and other suitablebinary constitutive expression vectors and tested in N. benthamiana.These include the genes encoding Sorbi-WRL1 (from Sorghum bicolor; SEQID NO:334), Lupan-WRL1 (from Lupinus angustifolius; SEQ ID NO:335) andRicco-WRL1 (from Ricinus communis; SEQ ID NO:336). These constructs aretested in comparison with the Arabidopsis WRI1-encoding gene in the N.benthamiana leaf assay.

As an initial step in the procedure, a partial cDNA fragmentcorresponding to the WRL1 was identified in the developing seed ESTdatabase of Lupinus angustifolius (NA-080818_Plate14f06.b1, SEQ IDNO:277). A full-length cDNA (SEQ ID NO:278) was subsequently recoveredby performing 5′- and 3′-RACE PCR using nested primers and cDNAsisolated from developing seeds of Lupinus angustifolius. The full lengthcDNA was 1729 bp long, including a 1284 bp protein coding sequenceencoding a predicted polypeptide of 428 amino acids (SEQ ID NO:337). Theentire coding region of the full length lupin WRL1 cDNA was then PCRamplified using forward and reverse primers which both incorporatedEcoRI restriction sites to facilitate the cloning into the pJP3343vector under the control of a 35S promoter in the sense orientation. A.tumefaciens strain AGL1 harbouring the pJP3343-LuangWRL1 was infiltratedin N. benthamiana leave tissues as described in Example 1. Leaf discstransiently expressing the pJP3343-LuangWRL1 were then harvested andanalysed for oil content.

Example 24 Silencing of the CGI-58 Homologue in N. tabacum

James et al. (2010) have reported that the silencing of the A. thalianaCGI-58 homologue resulted in up to 10-fold TAG accumulation in leaves,mainly as lipid droplets in the cytosol. Galactolipid levels were alsofound to be higher, whereas levels of most major phospholipid speciesremained unchanged. Interestingly, TAG levels in seeds were unaffectedand, unlike other TAG degradation mutants, no negative effect on seedgermination was observed.

Three full length and two partial transcripts were found in the N.benthamiana transcriptome showing homology to the A. thaliana CGI-58gene. A 434 bp region present in all five transcripts was amplified fromN. benthamiana isolated leaf RNA and cloned via LR cloning (Gateway)into the pHELLSGATE12 destination vector. The resulting expressionvector designated pTV46 encodes a hairpin RNA (dsRNA) molecule forreducing expression of the tobacco gene encoding the homologue of CG1-58and was used to transform N. tabacum as described in Example 1, yielding52 primary transformants.

Primary transformants displaying increased TAG levels in theirvegetative tissues are crossed with homozygous lines described inExample 20.

Example 25 Silencing of the N. tabacum ADP-Glucose Pyrophosphorylase(AGPase) Small Subunit

Sanjaya et al. (2011) demonstrated that silencing of the AGPase smallsubunit in combination with WRI over-expression further increases TAGaccumulation in A. thaliana seedlings while starch levels were reduced.An AGPase small subunit has been cloned from flower buds (Kwak et al.,2007). The deduced amino acid sequence showed 87% identity with the A.thaliana AGPase. A 593 bp fragment was synthesized and cloned intopHELLSGATE12 via LR cloning (Gateway) resulting in the binary vectorpTV35. Transformation of N. tabacum was done as described in Example 1and yielded 43 primary transformants.

Primary transformants displaying a reduction in total leaf starch levelsare crossed with homozygous lines described in Examples 20 and 21. Inaddition, primary transformants are crossed with homozygous lines thatare the result of a crossing of the lines described in 20 and 21.

Example 26 Production and Use of Constructs for Gene CombinationsIncluding an Inducible Promoter

Further genetic constructs are made using an inducible promoter systemto drive expression of at least one of the genes in the combinations ofgenes as described above, particularly in pJP3503 and pJP3502. In themodified constructs, the WRI1 gene is expressed by an inducible promotersuch as the Aspergillus niger alcA promoter in the presence of anexpressed Aspergillus niger alcR gene. Alternatively, a DGAT isexpressed using an inducible promoter. This is advantageous when maximalTAG accumulation is not desirable at all times during development. Aninducible promoter system or a developmentally-controlled promotersystem, preferably to drive the transcription factor such as WRI1,allows the induction of the high TAG phenotype at an appropriate timeduring development, and the subsequent accumulation of TAG to highlevels.

TAG can be further increased by the co-expression of transcriptionfactors including embryogenic transcription factors such as LEC2 or BABYBOOM (BBM, Srinivasan et al., 2007). These are expressed under controlof inducible promoters are described above and super-transformed ontransgenic lines or co-transformed with WRI and DGAT.

pJP3590 is generated by cloning a MAR spacer as a AatII fragment intothe AatII site of pORE04. pJP3591 is generated by cloning a second MARspacer as an KpnI fragment into the KpnI site of pJP3590. pJP3592 isgenerated by cloning the AsiSI-SmaI fragment of the DNA molecule havingthe nucleotide sequence shown in SEQ ID NO:416 (12ABFJYC_pJP3569_insert;FIG. 19B) into the AsiSI-EcoRV sites of pJP3591. pJP3596 is generated bycloning a PstI-flanked inducible expression cassette containing the alcApromoter expressing the M. musculus MGAT2 and a Glycine max lectinpolyadenylation signal into an introduced SbfI site in pJP3592.Hygromycin-resistant versions of both pJP3592 and pJP3596 (pJP3598 andpJP3597, respectively) are generated by replacing the NPTII selectablemarker gene with the HPH flanked gene at the FseI-AscI sites.

These constructs are used to transform the same plant species asdescribed in Example 20. Expression from the inducible promoter isincreased by treatment with the inducer of the transgenic plants afterthey have grown substantially, so that they accumulate increased levelsof TAG. These constructs are also super-transformed in stablytransformed constructs already containing an oil-increase constructincluding the three-gene or four-gene TDNA region (SEQ ID NO:411 and SEQID NO:412, respectively). Alternatively, the gene expression cassettesfrom the three-gene and four-gene constructs are cloned into the Nodsites of pJP3597 and pJP3598 to yield a combined constitutive andinducible vector system for high fatty acid and TAG synthesis,accumulation and storage.

In addition to other inducible promoters, an alternative is that geneexpression can be temporally and spatially restricted by using promotersthat are only active during specific developmental periods or inspecific tissues. Endogenous chemically inducible promoters are alsoused to limit expression to specific developmental windows.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

The present application claims priority from U.S. 61/580,590 filed 27Dec. 2011 and U.S. 61/718,563 filed 25 Oct. 2012, the entire contents ofboth of which are incorporated herein by reference.

All publications discussed and/or referenced herein are incorporatedherein in their entirety.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

REFERENCES

-   Abdullah et al. (1986) Biotech. 4:1087.-   Al-Mariri et al. (2002) Infect. Immun. 70:1915-1923.-   Alemanno et al. (2008) Planta 227:853-866.-   Almeida and Allshire (2005) TRENDS Cell Biol. 15:251-258.-   Alonso et al. (2010) Green Chem. 12:1493-1513.-   Alvarez et al. (2000) Theor. Appl. Genet. 100:319-327.-   Andrianov et al. (2010) Plant Biotech. J. 8:277-287.-   Barthole et al. (2011) Plant Sci. 185-186:33-39.-   Bartlett et al. (2008) Plant Methods 4:22.-   Baud et al. (2007) Plant J. 50:825-838.-   Baumlein et al. (1991) Mol. Gen. Genet. 225:459-467.-   Baumlein et al. (1992) Plant J. 2:233-239.-   Benghezal et al. (2007) J. Biol. Chem. 282:30845-30855.-   Bettany et al. (2003) Plant Cell Rep. 21:437-444.-   Bligh and Dyer (1959) Canadian Journal of Biochemistry and    Physiology 37:911-917.-   Bohner et al. (1999) Plant J. 19:87-95.-   Bourque (1995) Plant Sci. 105:125-149.-   Boutilier et al. (2002) Plant Cell 14:1737-1749.-   Bouvier-Nave et al. (2000) European Journal of Biochemistry/FEBS    267:85-96.-   Bower et al. (1996) Mol. Breed. 2: 239-249.-   Bradford (1976) Anal. Biochem. 72:248-254.-   Broothaerts et al. (2005) Nature 433:629-633.-   Broun et al. (1998) Plant J. 13:201-210.-   Bruce et al. (2000) P. Plant Cell 12:65-80.-   Buchanan-Wollaston (1994) Plant Physiol. 105:839-846.-   Busk et al. (1997) Plant J. 11:1285-1295.-   Cadwell and Joyce (1992) PCR Methods Appl. 2:28-33.-   Cao et al. (2003) J. Biol. Chem. 278:13860-13866.-   Cao et al. (2007) J. Lipid Res. 48:583-591.-   Capuano et al. (2007) Biotechnol. Adv. 25:203-206.-   Cernac and Benning (2004) Plant J. 40:575-585-   Chen et al. (2010) Biotechnol. for Biofuels. 3:9.-   Cheng et al. (1996) Plant Cell Rep. 15:653-657.-   Cheng et al. (2003) J. Biol. Chem. 278, 13611-13614.-   Chikwamba et al. (2003) Proc. Natl. Acad. Sci. U.S.A.    100:11127-11132.-   Christensen and Quail (1996) Transgenic Res. 5:213-218.-   Christie (1993) Advances in Lipid Methodology—Two, Oily Press,    Dundee, pages 195-213.-   Chung et al. (2006) BMC Genomics 7:120.-   Clough and Bent (1998) Plant J. 16:735-743.-   Coco et al. (2001) Nature Biotechnology 19:354-359.-   Coco et al. (2002) Nature Biotechnology 20:1246-1250.-   Comai et al. (2004) Plant J. 37: 778-786.-   Corrado and Karali (2009) Biotechnol. Adv. 27:733-743.-   Courvalin et al. (1995) Life Sci. 318:1207-1212.-   Coutu et al. (2007) Transgenic Res. 16:771-781.-   Crameri et al. (1998) Nature 391:288-291.-   Dalton et al. (1998) Plant Science 132:31-43.-   Dandik and Aksoy (1998) Fuel Process Technol. 57: 81-92.-   Darji et al. (1997) Cell 91:765-775.-   Dauk et al (2007) Plant Sci. 173:43-49.-   Deshpande (1992) Appl. Biochem. Biotechnol. 36:227-234.-   Dhadialla et al. (1998) Annu. Rev. Entomol. 43:545-569.-   Dietrich et al. (1998) Nature Biotech. 18:181-185.-   Dulermo and Nicaud (2011) Metab. Eng. 13:482-491.-   Durrett et al. (2008) Plant J. 54:593-607.-   Dyer et al. (2002) Plant Physiol. 130:2027-2038.-   Eggert et al. (2005) Chembiochem 6:1062-1067.-   Ellerstrom et al. (1996) Plant Mol. Biol. 32:1019-1027.-   Endalew et al. (2011) Biomass and Bioenergy 35:3787-3809.-   Felenbok (1991) J. Biotechnol. 17:11-17.-   Fennelly et al. (1999) J. Immunol. 162:1603-1610.-   Froissard et al. (2009) FEMS Yeast Res 9:428-438.-   Fujimura et al. (1985) Plant Tissue Culture Lett. 2:74.-   Gan (1995) Molecular characterization and genetic manipulation of    plant senescence. PhD thesis. University of Wisconsin, Madison.-   Gan and Amasino (1995) Science 270:1986-1988-   Gatz (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108.-   Ghosal et al. (2007) Biochimica et Biophysica Acta 1771:1457-1463.-   Ghosh et al. (2009) Plant Physiol. 151:869-881.-   Girgi et al. (2002) Molecular Breeding 10:243-252.-   Glevin et al. (2003) Microbiol. Mol. Biol. Rev. 67:16-37.-   Goffman et al. (2005) Plant Physiol. 138:2269-2279.-   Gong and Jiang (2011) Biotechnol. Lett. 33:1269-1284.-   Gould et al. (1991) Plant Physiol. 95:426-434.-   Grant et al. (1995) Plant Cell Rep. 15:254-258.-   Greenwell et al. (2010) J. R. Soc. Interface 7:703-726.-   Grillot-Courvalin et al. (1999) Curr. Opin. Biotech. 10(5):477-481.-   Grillot-Courvalin et al. (1998) Nature Biotech. 16:862-866.-   Gurel et al. (2009) Plant Cell Rep. 28:429-444.-   Harayama (1998) Trends Biotechnol. 16: 76-82.-   Haseloff and Gerlach (1988) Nature 334:585-591.-   Hellinga (1997) Proc. Natl. Acad. Sci. 94:10015-10017.-   Henikoff et al. (2004) Plant Physiol 135: 630-636.-   Hense et al. (2001) Cell Microbiol. 3:599-609.-   Hershey and Stoner (1991) Plant Mol. Biol. 17:679-690.-   Hinchee et al. (1988) Biotechnology 6:915-922.-   Hirayama and Hujii (1965) Agricultural and Biological Chemistry    29:1-6.-   Horvath et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97:1914-1919.-   Huang (1996) Plant Physiol. 110:1055-1061.-   Iwabuchi et al. (2003) J. Biol. Chem. 278:4603-4610.-   Izawati et al. (2009) J. Oil Palm Res. 21:643-652.-   James et al. (2010) Proc. Natl. Acad. Sci. USA 107:17833-17838.-   Jepson et al. (1994) Plant Mol. Biol. 26:1855-1866.-   Jézéquel et al. (2008) Biotechniques 45:523-532.-   Joviet et al. (2004) Plant Physiol. Biochem. 42:501-509.-   Karmakar et al. (2010) Bioresource Technology 101:7201-7210.-   Kindle (1990) Proc. Nat. Acad. Sci. USA 87: 1228-1232.-   Klein et al. (1998) Exp. Neurol. 150:183-194.-   Knothe (2005) Fuel Process. Technol. 86:1059-1070.-   Knothe and Steidley (2005) Fuel 84:1059-1065.-   Koziel et al. (1996) Plant Mol. Biol. 32:393-405.-   Kuhn et al. (2009) J. Biol. Chem. 284:34092-102.-   Kunik et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98:1871-1876.-   Kwak et al. (2007) Plant Physiol. 145:277-289.-   Lacroix et al. (2008) Proc. Natl. Acad. Sci. U.S.A. 105:    15429-15434.-   Lakshminarayana et al. (1984) JAOCS 61:1249-1253.-   Lam et al. (2010) Biotechnol. Adv. 28:500-18.-   Lardizabal et al. (2001) J. Biol. Chem. 276:38862-38869.-   Lardizabal et al. (2008) Plant Physiol. 148: 89-96.-   Larkin et al. (1996) Transgenic Res. 5:325-335.-   Lee et al. (1998) Science 280:915-918.-   Leung et al. (1989) Technique 1:11-15.-   Li et al. (1996) FEBS Lett. 379:117-121.-   Li et al. (2006) Phytochemistry 67: 904-915.-   Li et al. (2007) Proc. Natl. Acad. Sci. USA. 104:18339-44.-   Lin et al. (2005) Plant Physiol. Biochem. 43:770-776.-   Lindsey and Gallois (1990) Journal of Experimental Botany    41:529-536.-   Liu et al. (2010a) Fuel 89:2735-2740.-   Liu et al. (2010b) Plant Physiol. Biochem. 48: 9-15.-   Lui et al. (2009) J. Agric. Food Chem. 57: 2308-2313.-   Maher and Bressler (2007) Bioresource Technology 98:2351-2368.-   Mann et al. (2011) BMC Biotechnol. 11:74.-   Martinez and Jepson, Ecdysteroid agonist-inducible control of gene    expression in plants. In Inducible Gene Expression in Plants. Edited    by Reynolds PHS. New York: CABI Publishing; 1999:23-41.-   Martinez et al. (1999) Plant J. 19:97-106.-   Matsuoka et al. (1994) Plant J. 6:311-319.-   Meier et al. (1997) FEBS Lett. 415:91-95.-   Mett et al. (1993) Proc. Natl. Acad. Sci. USA 90:4567-4571.-   Millar and Waterhouse (2005). Funct Integr Genomics 5:129-135.-   Murashige and Skoog (1962) Physiologia Plantarum 15:473-497.-   Needleman and Wunsch (1970) J. Mol Biol. 45: 443-453-   Ness et al. (2002) Nature Biotechnology 20:1251-1255.-   Niedz et al. (1995) Plant Cell Reports 14:403-406.-   Ostermeier et al. (1999) Nature Biotechnology 17:1205-1209.-   Ow et al. (1986) Science 234:856-859.-   Padidam (2003) Curr. Opin. Plant Biol. 6:169-77.-   Padidam et al. (2003) Transgenic Res. 12:101-9.-   Panekina (1978) Chemistry of Natural Compounds 14:33-36.-   Parthibane et al. (2012) J. Biol. Chem. 287:1946-54.-   Pasquinelli et al. (2005). Curr Opin Genet Develop 15:200-205.-   Perez-Vich et al. (1998) JAOCS 75:547-555-   Perriman et al. (1992) Gene 113:157-163.-   Perrin et al. (2000) Mol Breed 6:345-352.-   Perry and Harwood (1993) Phytochemistry 33:329-333.-   Phillips et al. (2002) Journal of Food Composition and Analysis    12:123-142.-   Picard (1994) Cur. Top. Biotech. 5:511-515.-   Pigeaire et al. (1997) Mol. Breed. 3:341-349.-   Potenza et al. (2004) In Vitro Cell Dev. Biol. Plant 40:1-22.-   Powell et al. (1996) Vaccines 183, Abstract.-   Prasher et al. (1985) Biochem. Biophys. Res. Commun. 127:31-36.-   Qiu et al. (2001) J. Biol. Chem. 276:31561-3156-   Ramamoorthy and Kumar (2012) Plant Cell Rep. 31:1923-1931.-   Riddiford et al. (2000) Vitam. Horm. 60:1-73.-   Rossell and Pritchard (1991) Analysis of Oilseeds, Fats and Fatty    Foods. Elsevier-   Ruuska et al. (2002) Plant Cell 14:1191-1206.-   Ryan et al. (1984) J. Am. Oil Chem. Soc. 61:1610-1619.-   Saha et al. (2006) Plant Physiol. 141:1533-1543.-   Sanjaya et al. (2011) Plant Biotech. J. 9:874-883.-   Schaffner (1980) Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167.-   Science Publishers Ltd: London (Chapter 2, pp. 48-53).-   Scott et al. (2010) Plant Biotechnol. J. 8(8):912-27-   Semwal et al. (2011) Bioresource Technology 102:2151-2161.-   Senior (1998) Biotech. Genet. Engin. Revs. 15:79-119.-   Shiau et al. (2001) Vaccine 19:3947-3956.-   Shiina et al. (1997) Plant Physiol. 115:477-483.-   Shimada and Hara-Nishimura (2010) Biol. Pharm. Bull. 33:360-363.-   Shippy et al., (1999) Mol. Biotech. 12:117-129.-   Sieber et al. (2001) Nature Biotechnology 19:456-460.-   Siloto et al. (2009) Lipids 44:963-973.-   Sizemore et al. (1995) Science 270:299-302.-   Slade and Knauf (2005) Transgenic Res. 14: 109-115.-   Smith et al. (2000) Nature 407:319-320.-   Srinivasan et al. (2007) Planta 225:341-51.-   Stalker et al. 1988 Science 242: 419-423.-   Stemmer (1994a) Proc. Natl. Acad. Sci. USA 91:10747-10751.-   Stemmer (1994b) Nature 370(6488):389-391.-   Stobart et al. (1997) Planta 203:58-66.-   Tan et al. (2011) Plant Physiol. 156:1577-1588.-   Taylor (1997) The Plant Cell 9:1245-1249.-   Thillet et al. (1988) J. Biol. Chem 263:12500-12508.-   Tingay et al. (1997) Plant J. 11:1369-1376.-   Toriyama et al. (1986) Theor. Appl. Genet. 73:16-19.-   Tumaney et al. (2001) J. Biol. Chem. 276:10847-10852.-   Tzfira and Citovsky (2006) Curr. Opin. Biotech. 17:147-154.-   Ulmasov et al. (1995) Plant Physiol. 108:919-927.-   Unger et al. (2002) Transgenic Res. 11:455-465.-   van de Loo et al. (1995) Proc Natl Acad Sci USA. 92:6743-6747.-   Voinnet et al. (2003) Plant J. 33:949-956.-   Volkov et al. (1999) Nucleic Acids Research 27(18):e18.-   Wang et al. (2011) J. Agric. Food Chem. 60:144-52.-   Waterhouse et al. (1998). Proc. Natl. Acad. Sci. USA 95:13959-13964.-   Weinmann et al. (1994) Plant J. 5:559-569.-   Weiss (2003) Int. J. Med. Microbiol. 293:95-106.-   Weselake et al. (2009) Biotechnology Advances 27:866-878.-   Winans et al. (1988) Journal of Bacteriology 170:4047-4054.-   Wood et al. (2009). Plant Biotech. J. 7: 914-924.-   Wood et al., manuscript in preparation-   Yang et al. (2003) Planta 216:597-603.-   Yang et al. (2010) PNAS 107:12040-12045.-   Yen and Farese (2003) J. Biol. Chem. 278:18532-18537.-   Yen et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99:8512-8517.-   Yen et al. (2005) J. Lipid Res. 46: 1502-1511.-   Zhang et al. (1999a) Plant Cell Reports 18:959-966.-   Zhang et al. (1999b) Plant Cell, Tissue and Organ Culture 56:37-46.-   Zhao et al. (1998) Nature Biotechnology 16:258-261.-   Zheng et al. (2003) The Plant Cell 15:1872-1887.-   Zolotukhin et al. (1996) J. Virol. 70:4646-4654.

The invention claimed is:
 1. A process for producing an industrialproduct, the process comprising the steps of: i) obtaining a vegetativeplant part from a transformed plant having a total non-polar lipidcontent of at least 10% (w/w dry weight), ii) either a) converting atleast some of the lipid in the vegetative plant part of step i) to theindustrial product by applying heat, chemical, or enzymatic means, orany combination thereof, to the lipid in situ in the vegetative plantpart, or b) physically processing the vegetative plant part of step i),and subsequently or simultaneously converting at least some of the lipidin the processed vegetative plant part to the industrial product byapplying heat, chemical, or enzymatic means, or any combination thereof,to the lipid in the processed vegetative plant part, and iii) recoveringthe industrial product, thereby producing the industrial product.
 2. Theprocess of claim 1, wherein the step of physically processing thevegetative plant part comprises one or more of rolling, pressing,crushing or grinding the vegetative plant part.
 3. The process of claim1, which prior to step ii) further comprises the steps of: (a)extracting at least some of the non-polar lipid content from thevegetative plant part as non-polar lipid, and (b) recovering theextracted non-polar lipid.
 4. The process of claim 3, wherein (i) theextracted non-polar lipid comprises triacylglycerols, wherein thetriacylglycerols comprise at least 90% (w/w) of the extracted non-polarlipid, and/or (ii) the extracted non-polar lipid comprises free sterols,steroyl esters, steroyl glycosides, waxes or wax esters, or anycombination thereof.
 5. The process of claim 3, wherein step (a) uses anorganic solvent.
 6. The process of claim 3 which comprises one or moreof a) recovering the extracted non-polar lipid by collecting it in acontainer, b) one or more of degumming, deodorising, decolourising,drying or fractionating the extracted non-polar lipid, c) removing atleast some waxes and/or wax esters from the extracted non-polar lipid,and d) analysing the fatty acid composition of the extracted non-polarlipid.
 7. The process of claim 3 in which the volume of the extractednon-polar lipid is at least 1 liter.
 8. The process of claim 1, whereinthe industrial product is a hydrocarbon product.
 9. The process of claim1, wherein the vegetative plant part is an aerial plant part or a greenplant part.
 10. The process of claim 9, wherein the vegetative plantpart is a plant leaf or stem.
 11. The process of claim 1, furthercomprising a step of harvesting the vegetative plant part from a plantwith a mechanical harvester.
 12. The process of claim 11, wherein thevegetative plant part is harvested from the plant some time betweenabout the time of flowering of the plant to about the time senescence ofthe plant has started.
 13. The process of claim 1, wherein thevegetative plant part comprises a total non-polar lipid content of atleast about 15% (w/w dry weight).
 14. The process of claim 1, whereinthe vegetative plant part comprises a total TAG content of at leastabout 11% (w/w dry weight).
 15. The process of claim 1, wherein thevegetative plant part has one or more or all of the following features:i) oleic acid comprises at least 19% of the total fatty acid content inthe non-polar lipid in the vegetative plant part, ii) palmitic acidcomprises at least 20% of the total fatty acid content in the non-polarlipid in the vegetative plant part, iii) linoleic acid comprises atleast 15% of the total fatty acid content in the non-polar lipid in thevegetative plant part, and iv) α-linolenic acid comprises less than 15%of the total fatty acid content in the non-polar lipid in the vegetativeplant part.
 16. The process of claim 1, in which at least some of thelipid is converted to the industrial product by chemical means, whereini) the chemical means comprises reacting the non-polar lipid with analcohol, optionally in the presence of a catalyst, to produce alkylesters, and ii) optionally, blending the alkyl esters of step i) withpetroleum based fuel.
 17. The process of claim 16, wherein the alkylesters are methyl esters.
 18. The process of claim 1, wherein theindustrial product is synthetic diesel fuel and the process comprises:i) converting the non-polar lipid in the vegetative plant part, or theprocessed vegetative plant part, to a syngas by gasification, and ii)converting the syngas to the synthetic diesel fuel in a processcomprising using a metal catalyst or a microbial catalyst.
 19. Theprocess of claim 1, wherein the industrial product is a biofuel which isbio-oil or biogas, and the process comprises converting the non-polarlipid in the vegetative plant part or the processed vegetative plantpart to the bio-oil by pyrolysis, or to the biogas by gasification oranaerobic digestion.
 20. The process of claim 8, wherein the hydrocarbonproduct is a fatty acid ester, an alkane, a mixture of longer chainalkanes, an alkene, a biofuel, a bioalcohol, biochar, or a combinationof biochar, hydrogen and carbon monoxide.
 21. The process of claim 20,wherein i) if the hydrocarbon product is a fatty acid ester, then thefatty acid ester is a fatty acid methyl ester or a fatty acid ethylester; ii) if the hydrocarbon product is an alkane, then the alkane ismethane, ethane or a longer-chain alkane; and iii) if the hydrocarbonproduct is a bioalcohol, then the bioalcohol is an ethanol, propanol, orbutanol.